Correction of Preeclampsia by Intraplacental Gene Transfer of IGF-1 in the BPH/5 Mouse via NF-KB Mediated Induction of Angiogenic Gene Expression

Correction of Preeclampsia by Intraplacental Gene Transfer of IGF-1 in the BPH/5 Mouse via NF-KB Mediated Induction of Angiogenic Gene Expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Correction of Preeclampsia by Intraplacental Gene Transfer of IGF-1 in the BPH/5 Mouse via NF-KB Mediated Induction of Angiogenic Gene Expression Timothy Crombleholme, Kristen Moriarty, Sanjukta Majumder, Chia-Ling Kuo, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8563004/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Introduction. Preeclampsia causes severe complications for the mother, fetus, and newborn, yet the underlying mechanisms remain poorly understood. The BPH/5 mouse is the only spontaneous mouse model that recapitulates key features of human preeclampsia. Although impaired angiogenesis and endothelial dysfunction are hallmarks of this disease, the molecular pathways capable of restoring placental vascular integrity remain undefined. Existing animal models of preeclampsia have not directly targeted the placenta. We hypothesized that hIGF-1 rescues placental endothelial function by driving angiogenic gene expression through the IKK-β/NF-κB signaling axis, thereby correcting the maternal pathophysiologic features of preeclampsia. Methods. Placental morphometric analysis for CD31 immunostaining was performed on human placental samples from early onset preeclampsia (EOPE), gestational age-matched preterm premature rupture of membranes (PPROM), and term healthy controls. Next, primary human placental microvascular endothelial cells were cultured to evaluate IGF-1–mediated responses using in vitro angiogenesis assays under normoxic and hypoxic conditions, in the presence or absence of established NF-κB inhibitors. Cell proliferation was assessed using Ki-67 immunostaining and flow cytometry, and PCR-chromatin immunoprecipitation was used to quantify NF-κB binding to promoter regions of angiogenic genes in human placenta vascular endothelial cells as well as BeWo cells. In parallel, BPH/5 and C57BL/6 mice were time-mated and habituated to blood pressure cuff monitoring. Intraplacental gene delivery of 1x10 8 PFU of Ad-hIGF-1 (referred to as Ad-IGF-1) or Ad-LacZ was performed on embryonic day 16 with cerclage, followed by harvest on e21. Maternal endpoints of blood pressure and proteinuria were assessed at non-pregnant, first-trimester, pre-injection, and post-injection time points. Kidney histology, sFlt-1 levels, and placental endothelial microvascular density assessment were evaluated. Fetal endpoints included litter outcomes. Results. Morphometric placental analysis of EOPE vs. term healthy and PPROM controls showed that microvascular density is markedly reduced while villous architecture remains preserved. BPH/5 placentas similarly exhibit reduced microvascular density in comparison to C57BL/6 placentas. Restoration of microvasculature was appreciated after IGF-1 gene transfer. Angiogenesis and proliferation assays in HPVECs demonstrated that IGF-1 robustly enhances both angiogenic activity and cell proliferation under normoxic and hypoxic conditions, primarily through IKKβ/NF-κB–dependent transcriptional activation of key angiogenic genes. Interestingly, IGF-1 was found to enhance NF-κB signal transduction of angiogenic gene promoters in BeWo cells, but not HPVECs. In the BPH/5 mouse, intraplacental gene transfer of IGF-1 reduced the post-injection systolic, diastolic, and mean arterial pressures comparable to C57BL/6 controls, with the SBP consistently reduced at all delta comparisons across timepoints. Urinary protein levels in BPH/5 were also comparable to controls after gene transfer with Ad-IGF-1. Litter size, demise rate, reabsorptions, and pup weight were unaffected by Ad-IGF-1 gene transfer. Ad-IGF-1 treatment reduced glomerulosclerosis (47.2% vs. surgical sham; 58% vs. Ad-LacZ controls) while liver histology and s-Flt-1 were unchanged. Conclusion. IGF-1 gene transfer reverses the preeclampsia-like phenotype in BPH/5 mice by restoring placental microvascular density without affecting fetal outcomes in the first animal model of preeclampsia treatment that directly targets the placenta. Furthermore, IGF-1’s pro-angiogenic effects are suggested to occur via IKKβ/NF-κB–dependent activation. Health sciences/Diseases/Reproductive disorders Biological sciences/Biotechnology/Gene therapy Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Hypertension/Pre-eclampsia Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Preeclampsia has a major health impact on the maternal-fetal dyad and contributes to 5-15% of maternal deaths worldwide. 1 Preeclampsia is associated with increased fetal and perinatal morbidity and mortality, including fetal growth restriction 2 , preterm birth 3 , and higher rates of stillbirth 4 and neonatal complications 5 . Furthermore, rates of preeclampsia in the United States have increased by 25% from 1987 to 2004 2 . This disorder poses a substantial economic burden, with healthcare costs estimated at 2.18 billion dollars within the first year postpartum. 2 Despite its considerable morbidity, mortality, and financial impact, the pathophysiology of preeclampsia is poorly understood, and preterm delivery remains the sole definitive treatment. Low-dose aspirin is recommended as a preventive therapy in high-risk pregnancies beginning at 12 weeks of gestation; however, its effectiveness is modest. 6 Clinically, preeclampsia has been linked to maternal multi-organ failure, coagulopathic processes, and, in fetuses, can adversely affect fetal growth, leading to growth restriction. 2 The diagnosis in humans is established with the development of new-onset hypertension, defined as systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg, in combination with proteinuria (>300 mg per 24 hours). Preeclampsia with severe features is defined by either severe-range hypertension or evidence of end-organ injury, such as elevated liver enzymes, serum creatinine >1.1 mg/dL, thrombocytopenia (<100,000/µL), persistent headache, visual disturbances, or pulmonary edema. 2 The onset of preeclampsia is typically after 20 weeks of gestation, although earlier presentations can occur and are often associated with more severe maternal and fetal outcomes. Early onset preeclampsia (EOPE) is often associated with severe fetal growth restriction (FGR) and is defined as early onset preeclampsia (EOPE) when presenting less than 34 weeks. 7 The pathophysiology of preeclampsia is thought to be multifactorial, with key changes centering around antiangiogenic and inflammatory factors produced by the hypoxic placenta, likely secondary to aberrant spiral artery remodeling. 8,9 To date, animal models of preeclampsia have primarily focused on maternal treatment, with no models specifically targeting the placenta. 10-12 Previous studies have focused on diagnostic biomarker changes leading to the development of immune, vascular endothelial injury, and oxidative stress models of preeclampsia. 11 Prior research has suggested that the pathophysiology of preeclampsia involves dysregulated vascular tone mediated by VEGF and placental growth factor (PlGF). 13 These pro-angiogenic factors are antagonized in early pregnancy by excessive production of sFlt-1, a splice variant of VEGF, and later in gestation by sEng. 9 Although numerous molecular markers have been implicated in the pathophysiology of preeclampsia, a knowledge gap remains regarding how these factors interact to explain the overarching mechanisms driving this lethal disorder. Multiple mechanistic pathways likely contribute to disease onset, a concept supported by the more severe, early-onset forms of preeclampsia that are frequently associated with FGR. 7 Scant research has been performed on other key known markers, like insulin-like growth factor pathway (IGF-1), and its mechanistic implications for the development of preeclampsia, especially in EOPE. 14-16 Endothelial function in the placenta is regulated by trophoblast-derived angiogenic mediators such as VEGF, PlGF, and IGF-1, secreted primarily by syncytiotrophoblasts and cytotrophoblasts 17 . Among these, IGF-1 plays a crucial role in the physiology of endothelial cells by enabling proliferation, migration, angiogenesis, and the production of vasodilator nitric oxide and VEGF. 18-25 IGF is notably deficient in preeclampsia and may be a more specific biomarker for diagnosing preeclampsia than sFlt-1. 26 Mice with null mutations in IGF-1 or IGF-2 have a 60% reduction in birth weight compared to wild-type mice, and deficient IGF-1 also prevents VEGF-induced endothelial cell proliferation and survival. 27 We have previously demonstrated that the intraplacental gene transfer of IGF-1 corrects FGR in a naturally occurring rabbit model of fetal growth restriction 28 , as well as in the mesenteric uterine artery ligation model in mice. 29 The effects of IGF-1 in FGR may inform applications in other diseases within the context of uteroplacental insufficiency, specifically in EOPE. While these studies suggest that IGF-1 may be a promising therapeutic candidate, the precise molecular pathways through which it exerts these beneficial effects remain poorly understood. IGF-1 promotes angiogenesis and inhibits inflammation through activation of the PI3K–AKT–IKK–NF-κB signaling pathway, facilitating tissue repair and wound healing. 30 While NF-κB broadly regulates inflammation, oxidative stress, and angiogenesis in tumor cells and lymphocytes, endothelial cells exhibit unique NF-κB–dependent mechanisms. 31-33 Endothelial cell–specific inhibition or deletion of IKK-β impairs angiogenesis during both fetal and postnatal lung development, leading to fetal growth restriction. 34-36 Consistent with these findings, we previously demonstrated that NF-κB modulation is reduced in the pulmonary artery endothelial cells of growth-restricted fetal sheep. 37 Together, these studies suggest a mechanistic link between IGF-1 signaling, NF-κB activation, and placental endothelial cell function. We hypothesize that IGF-1 enhances placental endothelial proliferation and angiogenesis mediated by the IKK-β/NF-κB signaling axis, which corrects the placental vascular dysfunction associated with preeclampsia. To test this hypothesis, we first examined human placentas from pregnancies with EOPE, term healthy, and gestational age-matched preterm premature rupture of membrane (PPROM) controls to determine whether microvascular density is reduced in association with endothelial dysfunction. We used a spontaneous murine model of preeclampsia, the BPH/5 mouse, to determine whether this model recapitulates features of human placental pathology. The BPH/5 line, an inbred derivative of the hypertensive BPH/2 strain originally characterized by Davisson et al., 38 exhibits chronic hypertension and obesity. During pregnancy, the BPH/5 mouse recapitulates key features of human preeclampsia, including impaired trophoblast invasion, inadequate spiral artery remodeling, hypertension, proteinuria, glomerular endotheliosis, and systemic endothelial dysfunction. 39 We compared placental microvascular density between BPH/5 and normotensive C57BL/6 controls to determine the extent to which the murine model reflects the vascular deficiencies seen in human disease. To further delineate the mechanistic relationship between IGF-1 and NF-κB signaling, we performed angiogenesis and cell proliferation assays in placental endothelial cells and chromatin immunoprecipitation (ChIP) in both endothelial and BeWo cells to assess whether IGF-1 directly modulates NF-κB transcriptional activity through IKK-β and to confirm that IGF-1-mediated angiogenesis is driven by NF-κB binding to promoter regions of select angiogenic genes. Finally, to evaluate therapeutic potential, we conducted intra-placental gene transfer of human IGF-1 (hIGF-1) in pregnant BPH/5 mice and assessed maternal blood pressure, proteinuria, renal and hepatic histopathology, and placental microvascular structure. Through this integrative approach, we aimed to evaluate a causal role for impaired IGF-1/NF-κB signaling in the pathogenesis of preeclampsia and to demonstrate that restoration of placental IGF-1 expression corrects endothelial dysfunction and maternal disease. Results Demographics of Human Controls and Early-Onset Preeclampsia Participants Six healthy participants were recruited for placental assessment, with delivery occurring at a mean gestational age of 39 weeks 4 days ± 0 weeks 3 days. Among these, 4 of 6 were delivered via cesarean section and 2 of 6 via vaginal delivery. One patient with preterm premature rupture of membranes (PPROM) was recruited as a gestational age-matched control. She was diagnosed with PPROM at 30 weeks 4 days and delivered vaginally at 34 weeks 1 day. A total of five patients with early-onset preeclampsia were diagnosed at a mean gestational age of 31 weeks 6 days ± 2 weeks 2 days, with delivery at 32 weeks 5 days ± 2 weeks 4 days. Two of five had pre-existing chronic hypertension. Among these patients, 2 of 5 met criteria for severe preeclampsia based on transaminitis, 2 of 5 based on elevated blood pressures, and 1 of 5 based on features of a persistent headache. Delivery mode for the EOPE cohort was 3 cesarean sections and 2 vaginal deliveries. Lower Microvascular Morphometry in EOPE Placentas Compared with Healthy and PPROM Placentas EOPE placentas showed reduced histological CD31-positive microvascular lumens and disrupted vascular architecture compared with healthy controls, indicative of endothelial dysfunction (Fig. 1; immunofluorescence in an EOPE and early-onset fetal growth restriction [EOFGR] placenta). Morphometric analysis revealed that these microvascular alterations occurred with preservation of villous architecture. Microvascular density (MVD) was significantly lower in EOPE (Fig. 2), with a mean (95% CI) of 10.3 (6.4–14.2) microvessels per high-power field, compared with healthy controls [21.4 (17.5–25.3); p = 0.004] and PPROM [23.1 (14.4–31.8); p = 0.036], with no difference between healthy controls and PPROM. Lumens per villus were also significantly lower in EOPE [2.8 (1.4–4.1) lumens per villus] compared with healthy controls [5.3 (4.0–6.5); p = 0.034], with no significant difference between EOPE and PPROM [5.5 (2.4–8.6)], likely reflecting limited power. Total syncytial knot count (TSKC) was significantly higher in EOPE [11.7 (9.3–14.1) knots per 20× field] than in healthy [5.9 (3.8–8.1); p = 0.007] and PPROM [4.0 (-1.3–9.3); p = 0.036]. Chorionic villous counts and maximum and minimum villous diameters did not differ between groups, suggesting preserved villous architecture despite these microvascular changes. BPH/5 Placentas Also Exhibit Lower Microvascular Density, Restored by IGF-1 Gene Transfer Representative immunofluorescence (IF) staining for CD31 revealed disrupted microvascular architecture in BPH/5 surgical sham controls (Fig. 3a,b) compared to C57BL/6 surgical sham placentas. BPH/5 placentas treated with Ad-IGF-1 showed marked restoration of CD31-positive microvascular structures compared to BPH/5 Ad-LacZ controls (Figure 3c–f). Quantitative particle analysis confirmed that IGF-1 gene transfer significantly increased microvascular density (Fig. 3g, h), indicating improved placental vascularization in BPH/5 mice. BPH/5 mice treated with Ad-hIGF had significantly increased MVD with the estimated marginal mean (EMM) and standard error of 18.30 ± 0.87 compared to both BPH/5 Ad-LacZ (11.80 ± 0.88) and BPH/5 surgical sham (11.59 ± 0.97) (p < 0.001 for both). MVD values in BPH/5 after Ad-IGF-1 therapy were restored to levels similar to C57BL/6 surgical sham (16.65 ± 1.56), which showed no significant differences compared to C57BL/6 Ad-LacZ (16.60 ± 1.24) and C57BL/6 Ad-IGF-1 (19.05 ± 1.27). Collectively, these findings demonstrate that placental IGF-1 gene transfer normalizes MVD in BPH/5 mice, suggesting restoration of placental vascular integrity in the preeclamptic phenotype (Fig. 4). IGF-1 Restores Angiogenesis and Proliferation in Human Placental Microvascular Endothelial Cells Under Normoxia and Hypoxia To determine whether IGF-1 enhances endothelial function in the human placenta, we assessed angiogenic and proliferative responses in human placental microvascular endothelial cells (HPVECs) in normoxia (21% O₂) and hypoxia (5% O₂). In normoxia, IGF-1 treatment significantly increased tube formation, as reflected by a marked rise in angiogenic node formation compared with untreated controls. The extent of network restoration was comparable to the positive control, indicating that IGF-1 robustly promotes angiogenesis under normoxic conditions (Fig. 5A). In physiologic hypoxic placental conditions (5% FiO2), IGF-1 supplementation restored angiogenesis to levels equivalent to those observed in the positive control, demonstrating that IGF-1 effectively rescues endothelial function in conditions mimicking intrauterine hypoxia (Fig. 5B). Consistent with these findings, Ki-67 immunostaining revealed that IGF-1 significantly increased the proportion of proliferating HPVECs under both normoxia and hypoxia (Fig. 5C, D). Under each condition, the proliferative index in IGF-1–treated cells matched that of the positive control, confirming IGF-1’s potent proliferative effect on placental microvascular endothelium. Collectively, these results demonstrate that IGF-1 enhances both angiogenesis and proliferation in HPVECs in both normoxic and hypoxic conditions, supporting a role for IGF-1 in maintaining placental vascular integrity. IGF-1–Induced Angiogenesis and Proliferation Depend Primarily on Ikkβ/NF- k B Signaling To delineate the signaling mechanisms underlying IGF-1–mediated endothelial activation, HPVECs were treated with IGF-1 in the presence of pharmacologic inhibitors targeting IKKβ/NF-κB (BAY-11-7082), mTOR (rapamycin), PI3K (LY-294002), or Akt (GDC-0068). Under normoxic conditions, IGF-1 significantly enhanced tube formation, an effect abolished by IKKβ inhibition and partially reduced by rapamycin, suggesting contributions from both NF-κB and mTOR pathways. In contrast, PI3K and Akt blockade had minimal impact (Fig. 6A). Under hypoxia, IGF-1 induced a robust angiogenic response that was completely abolished by IKKβ inhibition and significantly reduced by rapamycin, while PI3K and Akt inhibitors modestly decreased tube formation. IGF-1 increased HPVEC proliferation under normoxia, which was significantly attenuated by IKKβ and, to a lesser extent, mTOR inhibition, but not by inhibitors of PI3K or Akt. Similarly, IGF-1–driven proliferation under hypoxia was markedly reduced by IKKβ inhibition and blunted by rapamycin. These findings identify IKKβ/NF-κB as an important mediator of IGF-1–induced angiogenic and proliferative responses in placental endothelial cells, with mTOR contributing as a secondary pathway. Together, they highlight NF-κB activation as an important signal transduction pathway by which IGF-1 preserves placental vascular function under stress (Fig. 6) IGF-1 Enhances NF-κB Recruitment to Angiogenic Gene Promoters To test whether IGF-1 directly promotes NF-κB–dependent transcription of angiogenic genes, we performed chromatin immunoprecipitation followed by qPCR (ChIP–qPCR) using an antibody against phospho-NF-κB p65 in both BeWo and HPVECs. Promoter occupancy was analyzed at promoter regions for vascular endothelial growth factor A ( VEGFA) , placental growth factor (PlGF), and hypoxia inducible factor 1 alpha ( HIF1a) genes in trophoblasts and placental microvascular endothelial cells under basal, lipopolysaccharide (LPS)-stimulated, and IGF-1–stimulated conditions. In BeWo cells, IGF-1 stimulation led to a pronounced enrichment of p65 at the VEGFA gene (~15-fold increase relative to control), whereas LPS produced only moderate (~5-fold) enrichment (Fig. 7A). At the PGF gene, IGF-1 induced a ~60-fold enrichment compared to ~5-fold for LPS (Fig. 7B). Similarly, p65 binding to the HIF1A increased ~25-fold following IGF-1 stimulation, in contrast to ~3-fold with LPS (Fig. 7C). In HPVECs, IGF-1 stimulation showed no difference in VEGFA, PlGF or HIF1A compared to unstimulated control (Fig. 8). These results suggest that IGF-1 selectively enhances NF-κB p65 recruitment to promoters of VEGF, PlGF, HIF-1a in BeWo cells but not HPVECs, distinguishing IGF-1–driven NF-κB activation from the classical inflammatory NF-κB response induced by LPS. This finding suggests a mechanistic link between IGF-1 signaling and transcriptional activation of placental angiogenesis pathways. The attenuation of angiogenesis in HPVECs with Bay-11-7082 compound suggests that NF-kB stimulates angiogenic gene expression, however the lack of binding to promoters of VEGFA, PlGF and HIF-1a suggests that other angiogenic genes may be responsible in HPVECs. IGF-1 Gene Transfer Normalized Systolic, Diastolic, And Mean Arterial Blood Pressures in the BPH/5 Mice Compared to C57BL/6 Controls Study groups: C57BL/6 Ad-LacZ treated (n=5), C57BL/6 Ad-IGF-1 treated (n=8), BPH/5 Ad-LacZ treated (n=5), and BPH/5 Ad-IGF-1 treated (n=6)—with observations recorded per animal across the different physiological time points (non-pregnant, 1st trimester (e5-e13), pre-injection (e14-e16), and post-treatment (e19-e21), as reflected in the summary data as follows. Systolic Blood Pressure (SBP): SBP was significantly higher in BPH/5 mice compared with C57BL/6 mice post-treatment with Ad-LacZ (157.5 ± 7.7 vs. 120.2 ± 7.7 mmHg; p = 0.0013) (Fig. 9a). Furthermore, injection with Ad-IGF-1 showed a lower SBP (124.3 mmHg) compared to injection with Ad-LacZ (157.5 mmHg) (p 0.05), whereas significant post-injection changes were observed following Ad-LacZ, with notable differences in changes between groups. In contrast, no significant differences in changes from any reference period to post-injection between Ad-IGF-1 and Ad-LacZ were observed in C57BL/6 mice (all p > 0.24) (Fig. 10a,b). Thus, Ad-IGF-1 therapy significantly attenuates systolic blood pressure in BPH/5 mice, resulting in lower post-injection SBP trajectories compared with the hypertensive response observed following Ad-LacZ treatment. Diastolic Blood Pressure (DBP): The mean DBP post-injection for BPH/5 Ad-LacZ mice was 123.2 mmHg compared to 92.0 mmHg in BPH/5 mice with Ad-IGF-1 (p=0.0158) (Fig. 9b). Compared to BPH/5 mice treated with Ad-LacZ, those treated with Ad-IGF-1 showed no significant post-injection changes across any reference period (p > 0.05). In contrast, Ad-LacZ-treated mice showed increased DBP consistently across reference periods, with a significant difference in the change from first trimester to post-injection between groups (post-injection – first trimester -4.33 in IGF vs. 35.1 in Ad-LacZ, p=0.025). In C57BL/6 mice, no significant differences in changes from any reference period to post-injection were observed between Ad-IGF-1 and Ad-LacZ (Fig. 10c,d). Mean Arterial Pressure (MAP): IGF treatment significantly lowered the MAP in BPH/5 mice compared to those treated with Ad-LacZ (p = 0.0026), normalizing it to levels comparable with C57BL/6-Ad-IGF-1 treated mice (p = 0.786) (Fig. 9C). Similarly, in BPH/5 mice, those treated with Ad-LacZ and Ad-IGF-1 showed significant differences in the non-pregnant to post-injection change (post-injection – non-pregnant -4.48 in Ad-IGF-1 vs. 26.54 in Ad-LacZ) and the first trimester to post-injection change (post-injection – first trimester 5.53 in Ad-IGF-1 vs. 36.80 in Ad-LacZ). In C57BL/6 mice, no significant differences between Ad-IGF-1 and Ad-LacZ were detected in changes from the non-pregnant or first-trimester periods to post-injection (p = 0.725 and p = 0.887, respectively), whereas a modest difference was observed for the post – pre-injection 12.19 in Ad-IGF-1 vs. -20.95 in Ad-LacZ (p = 0.044) (Fig. 10e,f). Collectively, these findings indicate that Ad-IGF-1 treatment therapy improved systolic, diastolic, and mean arterial pressures following injection in BPH/5 mice compared with Ad-LacZ–treated controls. When blood pressure was analyzed longitudinally using post-treatment–referenced deltas, systolic hypertension emerged as the most affected parameter, remaining consistently elevated in the Ad-LacZ cohort across all comparisons, whereas effects on diastolic pressure and mean arterial pressure were more modest and less consistently statistically significant. Murine Proteinuria Is Reduced by Ad-hIGF Treatment In BPH/5 Mice Urinary protein was tracked across time at non-pregnant, first-trimester, pre-, and post-injection time points. Among the BPH/5 cohorts, estimated marginal means (EMMs) ± standard errors (SEs) were calculated for each strain with Ad-IGF-1 or Ad-LacZ using a linear mixed-effect model (Fig. 11). In the BPH/5 cohort, Ad-IGF-1 treatment reduced the post-injection change in urinary protein in comparison to the non-pregnant state (−0.44 ± 0.17) compared with Ad-LacZ (0.09 ± 0.16), with a significant change difference (ΔEMM = −0.53 ± 0.23; p = 0.039). No significant trends were seen for post-first trimester or post-pre-injection comparisons. Litter Size, Rate of Demise, Reabsorption, And Pup Weight Were Not Altered by Therapy With IGF-1 Ad-IGF-1 therapy does not affect live birth rates in BPH/5 mice (Figure 12a). The probability of live birth for BPH/5 Ad-IGF-1 was 0.29 (0.18-0.42), BPH/5 Ad-LacZ was 0.47 (0.34-0.61), and BPH/5 surgical sham was 0.50 (0.34-0.66), and pairwise comparisons showed no difference in live birth rate between groups. IGF-1 treatment does not affect the rate of pup demise in BPH/5 mice (Figure 12b), with the number of demises per litter being low across all experimental groups. In C57BL/6, surgical sham showed higher odds of demise than Ad-LacZ (OR 5.82, p < 0.001) and Ad-IGF-1 (OR 3.35, p = 0.032), whereas no significant difference was observed between Ad-LacZ and Ad-IGF-1 (p = 0.505). The number of resorptions per litter was generally low across all groups (Figure 12c). In BPH/5 mice, Ad-IGF-1-treated mice showed a higher probability of resorption than Ad-LacZ-treated mice (OR 10, p = 0.002), while the surgical sham group showed low resorption. In C57BL/6 mice, resorption rates were generally low, with higher odds in Ad-LacZ–treated mice compared with Ad-IGF-1-treated mice (OR = 12.5, p = 0.042). However, because the direction of the effect is opposite between strains, this trend is likely unreliable and requires further investigation. IGF-1 therapy did not improve pup weight in BPH/5 mice (Fig. 12d). No differences in pup weight were observed between Ad-IGF-1 and Ad-LacZ groups within either strain, whereas surgical sham pups weighed more than those from virally treated pregnancies. These differences may reflect physiological responses associated with targeted intraplacental delivery, independent of vector or transgene identity. IGF treatment reduces glomerulosclerosis in BPH/5 mice. The glomerulosclerosis index (GSI) was markedly elevated in BPH/5 mice compared with C57BL/6 controls, consistent with the increased renal pathology observed in the BPH/5 mice. Gene transfer with Ad-LacZ did not significantly alter GSI compared with the BPH/5 surgical sham, confirming it as a suitable control. The estimated marginal mean and standard error (EMM ± SE) glomerulosclerosis index (GSI) for BPH/5 Ad-LacZ mice was 232.20 ± 27.17, compared with 98.20 ± 21.48 in BPH/5 mice treated with Ad-IGF-1 (p=0.006), representing an approximately 58% reduction with IGF-1 treatment relative to those treated with Ad-LacZ and 47.2% to BPH/5 surgical shams. Consistent with these quantitative findings, Figs. 13a and 13b show clear evidence of improved glomerular architecture and reduced glomerulosclerosis following Ad-IGF-1 treatment. Note, the GSI in BPH/5 IGF-treated mice was not significantly different from C57BL/6-Ad-IGF-1 controls, indicating that IGF-1 restored glomerular histology toward baseline levels. Liver dysfunction does not accompany the BPH/5 model of pre ‑ eclampsia Liver macrosteatosis was assessed in C57BL/6 and BPH/5 mice under non-pregnant (NP), Ad-LacZ-treated, Ad-IGF-1-treated, and surgical sham conditions. Livers were graded from 0-3, with 0 representing no evidence of macrosteatosis and 3 representing global macrosteatosis. BPH/5 mice had higher levels of macrosteatosis in the non-pregnant state, estimated marginal mean and standard error (EMM ± SE) (2.40 ± 0.36) compared to BPH/5 surgical sham (0.75 ± 0.40), BPH/5 Ad-LacZ (0.70 ± 0.36), and BPH/5 Ad-IGF-1 (0.64 ± 0.31), p ≤ 0.034. No significant differences were observed among the treated or sham groups (all p > 0.99). These results indicate strain-specific elevation in liver measurements in BPH/5 mice, with no detectable effect of IGF-1 treatment (Fig. 14). IGF treatment does not alter Soluble Fms-like Tyrosine Kinase-1 (sFlt-1) sFlt-1 levels were measured in C57BL/6 and BPH/5 mice under baseline non-pregnant (NP) conditions and following Ad-LacZ or Ad-IGF-1 treatment. In C57BL/6 mice, baseline estimated marginal means and standard error for s-Flt were low (6,183 ± 1,943 pg/ml, n=6 mice) and increased in pregnancy as expected, but with no significant difference in sFlt-1 between treatments with Ad-LacZ (22,558 ± 2054, n=6) and Ad-IGF-1 (25,327 ± 4,095 pg/ml, n=7). In BPH/5 mice, baseline sFlt-1 was very low (3,379 ± 3896 pg/ml, n = 4) and rose in pregnancy with Ad-LacZ (25,755 ± 4498, n = 3) and Ad-IGF-1 treatment (26,090 ± 2945 pg/ml, n = 7). The difference between these treatments was minimal and not statistically significant (p = 0.998). Overall, IGF did not further alter sFlt-1 compared to controls within either strain. Methods Human Placental Collection. A prospective study enrolled patients diagnosed with EOPE, term healthy controls, and PPROM for placental collection from April 2024 to August 2025 following Institutional Review Board (IRB) approval from UConn Health (IRB# 24-099J-1). Informed consent was obtained from all patients. Placentas were harvested according to prior guidelines set out by Burton et al. 40 A placental biopsy, approximately 3 cm x 3 cm, was taken in a random lateral quadrant free from infarctions, with care to avoid the umbilical cord insertion. The placenta sample was further dissected, removing the decidual and chorionic plates to isolate the mid-placental surface for analysis. Samples were fixed in 10% neutral-buffered formalin for 24 hours and stored in 70% ethanol until paraffin embedding. The tissues were cryosectioned at 5 µm thickness and mounted onto Superfrost Plus glass slides for later staining. Microvascular Density Assessment in Human Placentas. Samples were stained for CD31 using the above immunofluorescence protocol with primary antibody for CD31 at 1:100 (Thermo Fisher, Waltham, MA, USA), followed by Goat Anti-Rabbit IgG (H+L) Alexa Fluor 488 Conjugated Secondary Antibody (Thermo Fisher) at 1:200. Blinded quantitative morphometric analysis was performed on hematoxylin and eosin–stained placental sections. Two sections per placenta were analyzed. Chorionic villus count (CVC) and maximal and minimal chorionic villus diameter were measured at 10× magnification in 5 randomly selected fields per section. Total syncytial knot count (TSKC) was assessed at 20× magnification in 5 randomly selected fields. Luminal count (LC) per villus and total microvascular density were quantified at 40× magnification in oil in 10 randomly selected regions per section. All measurements were performed in a blinded manner using digital image analysis software to ensure reproducibility and minimize observer bias. Samples were imaged using a Zeiss fluorescent microscope (Oberkochen, Germany). Immunofluorescence. Frozen tissue samples embedded in OCT compound were sectioned coronally to visualize all layers, including the maternal decidua, the junctional zone with spongiotrophoblasts and glycogen cells, the labyrinth zone where maternal and fetal blood spaces intermingle, and the fetal-facing chorionic plate containing fetal vessels. The tissues were cryosectioned at 5 µm thickness and mounted onto Superfrost Plus glass slides. Frozen sections stored at -80°C were warmed to room temperature and placed in (50%-100%) ethanol for 5 minutes at room temperature. Slides were then placed in a 1:1 methanol/acetone mixture at -20°C for 10 minutes. Slides were permeabilization and blocked for intracellular antigen detection for 1 hour using 30 ml PBS, 60 μl 0.1% Triton X-100, 60 μl Tween, 60 μl Triton. After blocking, slides were incubated overnight using a 1:100 dilution of Anti-mouse/rCD31 goat (Invitrogen, Waltham, MA, USA).). The next day, samples were washed with PBS and incubated for 1 hour in a 1:200 dilution of Alex 546 Donkey anti-Goat (Invitrogen). Tissue sections were incubated with DAPI (1 µg/mL in PBS) for 10 minutes at room temperature to stain nuclei, followed by a final PBS wash. Imaging - Samples were imaged using a Zeiss fluorescence microscope with filter sets appropriate for DAPI (excitation 358 nm, emission 461 nm) and Alexa Fluor 546 (excitation 556 nm, emission 573 nm). Murine Placental Microvascular Density Assessment. Fetal endothelial microvascular density (MVD, expressed as % area or normalized vessel coverage) was quantified in BPH/5 and C57BL/6 placentas, including surgical sham controls, Ad-LacZ, and Ad-hIGF treated groups, following immunohistochemical staining for CD31, an endothelial cell marker of placental vasculature. Images were acquired at 10× magnification in three to four representative regions per placenta using a Zeiss fluorescent microscope. Placental morphometric analysis was performed in ImageJ (NIH, Bethesda, MD, USA). 41 For image processing, red-channel images were converted to 8-bit, binarized, and holes were filled. The scale was uniformly set across all 10× images (distance in pixels = 1; known distance = 83.82; unit = µm). Particle analysis was performed with a size threshold of 25–µm² and circularity set to 1.0, following manual thresholding. Extracted parameters included vessel count, total vessel area, average vessel size, and % area. The tissue area of each 10× image was 1040 × 1388 pixels (1.013 × 10¹⁰ µm²). Microvascular density was calculated as total vessel area (µm²) divided by total tissue area (µm²). Human Placental Microvascular Endothelial Cell (HPVEC) Culture. Human Placental Microvascular Endothelial Cells (HPVECs) were obtained from ScienceCell Laboratories (Carlsbad, CA, USA) and maintained in the manufacturer-recommended endothelial cell medium supplemented with growth factors and 5% CO₂ at 37°C under normoxia (21% O₂). Culture plates were pre-coated with fibronectin to promote cell adhesion. The culture medium was replaced every two days, and cells were passaged upon reaching 80–90% confluency. All experiments were performed using cells between passages 3 and 5 and in replicates (n ≥ 6). Reagents for Human In Vitro Studies and Chromatin Immunoprecipitation Assay . IGF-1 LR3 was sourced from PeproTech (Rocky Hill, NJ, USA). Pharmacologic inhibitors—such as BAY-11-7082 (IKK-β inhibitor), Rapamycin (mTOR inhibitor), GDC-0068 (pan-AKT inhibitor), and LY-294002 (pan-PI3K inhibitor)—came from MedChemExpress (Monmouth Junction, NJ, USA). The Ki67-PE antibody for flow cytometry was obtained from BD Biosciences (San Jose, CA, USA). Phospho-NF-κB p65 and the SimpleChIP® Enzymatic Chromatin IP Kit for chromatin immunoprecipitation assay were procured from Cell Signaling Technology (Danvers, MA). For RNA isolation, cDNA synthesis, and RTqPCR analyses, the RNeasy Mini Kit was purchased from Qiagen (Germantown, MD, USA), and the iScript™ gDNA Clear cDNA Synthesis Kit, iTaq™ Universal SYBR® Green Supermix, were obtained from Bio-Rad (Hercules, CA, USA). All primers, including mouse Igf1 and human Vegfa , Pgf , and Hif1a, were obtained from Bio-Rad (Hercules, CA, USA). In Vitro Angiogenesis Assay. Human placental microvascular endothelial cells (HPVECs; ScienceCell) were seeded on Geltrex™ basement membrane matrix (Thermo Fisher Scientific) to assess angiogenesis. Ninety-six–well plates were coated with 50 µL of Geltrex and incubated at 37 °C for 30 min to allow solidification. Cells (2.5 × 10⁴ per well) were cultured in complete or incomplete endothelial medium supplemented with recombinant human IGF-1 (100, 250 or 500 ng mL⁻¹). IGF-1 at 250 and 500 ng mL⁻¹ enhanced tube formation; 500 ng mL⁻¹ was used for subsequent assays. To evaluate pathway involvement, cells were then co-treated with IGF-1 (500 ng mL⁻¹) and one of four pharmacologic inhibitors—BAY-11-7082, rapamycin, GDC-0068, or LY-294002—at their IC₅₀ concentrations. Tube formation was monitored for 18 hours under normoxia (21% O₂) or hypoxia (5% O₂) using a controlled hypoxia chamber. Images were acquired on an inverted phase-contrast microscope, and tube networks were quantified using the Angiogenesis Analyzer plugin in ImageJ. 41 In Vitro Proliferation Assay. Cell proliferation was quantified by Ki-67 immunostaining and flow cytometry. HPVECs (2.5 × 10⁴ per well) were seeded in 24-well plates and maintained overnight in low-serum medium (2% FBS) to promote survival and synchronize cells in a quiescent state. The following day, cells were treated with complete medium (10% FBS), low-serum medium (2% FBS), IGF-1 (500 ng mL⁻¹), or IGF-1 (500 ng mL⁻¹) combined with one of the inhibitors (BAY-11-7082, rapamycin, GDC-0068, or LY-294002) at their IC₅₀ concentrations in low serum medium. Cultures were maintained for 48 h with medium replacement at 24 h under normoxic (21% O₂) or hypoxic (5% O₂) conditions. After treatment, cells were trypsinized, fixed by dropwise addition of ice-cold 70% ethanol, and stored at −20 °C for ≥ 2 h. Fixed cells were washed twice with BD staining buffer, resuspended in 100 µL buffer, and incubated with 10 µL PE-labelled anti–Ki-67 antibody (BD Biosciences) for 30 min at room temperature in the dark. After washing, samples were analyzed by flow cytometry. All experiments were performed in biological sextuplicates (n = 6). Chromatin immunoprecipitation and quantitative PCR (ChIP–qPCR) in BeWo and HPVECs. Chromatin immunoprecipitation was performed using the SimpleChIP® Enzymatic Chromatin IP Kit (magnetic beads; Cell Signaling Technology, Danvers, MA, USA)) according to the manufacturer’s protocol. BeWo as well as HPVECs were cross-linked with 1% formaldehyde for 10 min at room temperature, and the reaction was quenched with 125 mM glycine. Cells were washed with ice-cold PBS, collected, and lysed to isolate nuclei. Chromatin was digested with micrococcal nuclease and briefly sonicated to yield DNA fragments of 150–900 bp. Immunoprecipitation was carried out overnight at 4 °C using an antibody against phospho–NF-κB p65 (Cell Signaling Technology, Cat. #71254) or normal rabbit IgG (negative control). Immune complexes were captured with magnetic beads, washed sequentially, and eluted. Cross-links were reversed by incubation at 65 °C, and DNA was purified using spin columns provided in the kit. Purified DNA was analyzed by quantitative PCR using SYBR Green master mix (Bio-Rad, Hercules, CA, USA) with primer sets for VEGFA, PlGF, and Hif-1A (Bio-Rad). Fold enrichment was calculated as the percent input normalized to IgG controls. Reactions were performed in technical duplicates from three independent biological replicates. Animals . Virgin control C57BL/6 mice at 8-12 weeks of age were obtained from prior in-house colonies. Virgin BPH/5 mice were originally gifted from Dr. Jennifer Sones (Colorado State University) and maintained as an in-house colony. C57BL/6 mice were chosen as controls, as they have been used in previous BPH/5 studies and were used in the initial eight-way cross to derive the final BPH-5 strain, and are one of the most original relatives to the BPH/5 line. 38 The University of Connecticut Animal Care and Use Committee approved all experiments. Care of the mice met all standards set forth by the National Institute of Health (NIH) guidelines on the care and use of animals, regulations set forth by the United States Drug Association (USDA), and the Animal Veterinary Medical Association Panel Euthanasia. Adenoviral Constructs . The Ad-hIGF-1 adenoviral construct is a replication-defective Ad5 vector (E1/E3 deleted) under the control of the CMV promoter. Human IGF (h-IGF)-1 construct was generously supplied by L. Sweeney (University of Pennsylvania, Philadelphia, PA). The recombinant adenovirus carrying the β-galactosidase transgene (Ad-LacZ) was obtained from J. Wilson (University of Pennsylvania) and was also an Ad5 vector (E1/E3 deleted) under a CMV promoter. The parental adenovirus for Ad-hIGF-1 was Ad-Easy, and for Ad-LacZ was dl 7001. Viruses were prepared with 293 cells and purified and stored in 50% glycerol at -80C and viral titers were determined using absorbance at 260 nm and diluted 1:10 with phosphate-buffered saline (PBS) before use in the mice to prevent glycerol toxicity. Timed Mating. All animals were housed in a facility with constant temperature and humidity and under a controlled 12:12 h light/ dark cycle and allowed free access to standard chow and water. All mice (C57BL/6 and BPH/5) were time mated (based on Charles River breeding protocol: day 1 of gestation is defined as the observed day of vaginal plug after male and female mating). Blood Pressure Monitoring . BPH/5 mice have significantly elevated baseline MAP compared with control mice before pregnancy (128±5 versus 106±7 mm Hg, respectively; P<0.01). 38 MAP remains stable in BPH/5 and C57BL/6 mice throughout the first 2 weeks of pregnancy. 38 However, beginning in the last trimester (day 14), MAP begins to rise even further in BPH/5 mice, increasing to peak levels just before delivery. MAP returns to pre-pregnancy levels by postpartum day 2 or 3. In sharp contrast, MAP falls at the end of the second trimester in C57BL/6 mice for a short period but returns to pre-pregnancy levels several days before delivery, where it remains throughout the postpartum period. 38 C57BL/6 and BPH/5 mice were acclimated to blood pressure tail cuff monitoring before breeding. Blood pressure monitoring occurs as follows. All experimental mice were habituated to the CODA 8 non-invasive blood pressure system pre-pregnancy to allow habituation of tail cuff monitoring as described in prior habituation protocols. 42 Murine tail cuff monitoring was performed with the CODA 8 non-invasive blood pressure acquisition system (Kent Scientific, Torrington, CT), which uses VPR to detect blood pressure based on volume changes in the tail. The CODA system was set and calibrated with factory standards. Patency of the occlusion and VPR cuffs was checked routinely. The blood pressure measurements were conducted in a designated quiet area, and the CODA incubator was preheated to 37˚C to maintain body temperature during each measurement. Mice were encouraged to walk into the restraint tubes, and the tube end holders were adjusted to prevent excessive movement. The test clamp was placed at the end of each mouse tail, and the blood pressure was measured after the heartbeat was noted to have stabilized. To measure blood pressure, the occlusion cuff is inflated to 250 mm Hg and deflated over 20 s. The VPR sensor cuff detects changes in the tail volume as the blood returns to the tail during the occlusion cuff deflation. The minimum volume change was set as 15 μL. Each recording session consisted of 15 to 25 inflation and deflation cycles per set, of which the first 5 cycles were “acclimation” cycles and were not used in the analysis, whereas the following cycles were used. When possible, three measurements were recorded, and the average measurement was reported for that evaluation. Study Design for Animal Experimentation . For experiments, C57BL/6 and BPH/5 mice were time-mated, and blood pressure was recorded pre-pregnancy, in the first trimester (e5-e13), pre-injection (e14-e16), and post-injection at the time of harvest (e19-e21). Urine was collected at the same time points. To test the hypothesis that IGF-1 can treat the preeclampsia phenotype, a laparotomy was performed on e16, and both uterine horns were exposed. 1x10 8 plaque-forming units (pfu) of Ad-hIGF-1 or Ad-LacZ was administered to each placenta along with the placement of a permanent cerclage with silk. Final delivery was performed at embryonic day 21 (E21) via laparotomy, with collection of pups, placentas, and maternal liver and kidney tissues. Surgical sham BPH/5 and C57BL/6 mice underwent laparotomy on e16 with cerclage placement, with final harvest similarly at e21. At the time of harvest, fetal pup weights, live births, and the number of resorptions were noted. Placentas were frozen in OCT media for future cryo-sectioning and immunohistochemical analysis. Maternal liver and kidney were collected and paraffin-embedded. A cardiac needle aspiration was again performed for serological analysis of sFlt-1. Primary outcome measures were improvement in the preeclampsia phenotype through reduction in blood pressure and proteinuria. Secondary outcome data were fetal pup weight, litter size, reabsorption rate, sFLT-1 levels, glomerulosclerosis, hepatic macrosteatosis, and placental microvascular density of fetal vascular endothelial cells. Murine Urine Protein Assessment . Mice were restrained by gently grasping the base of the tail with the dominant hand and placing the thumb on either side of the neck with the non-dominant hand. The tail was then released while gently stroking the belly, and the animal was held over the collection device. We used 0.5-ml Eppendorf tubes to collect urine. Urine was frozen at −80°C until analysis for total protein content using the protein assay kit (Bio-Rad) according to the manufacturer’s instructions, which required 5 microliters of sample. Histology . Murine Liver and kidney tissues were fixed in 10% neutral-buffered formalin for 24 hours and stored in 70% ethanol until paraffin embedding. Paraffin blocks were sectioned at 5 µm and mounted on Superfrost Plus slides, which were baked to ensure adherence. Slides were deparaffinized using Histo-Clear followed by a graded ethanol series (100%–70%) and rinsed in deionized water. Liver sections were stained with hematoxylin and eosin (H&E) to evaluate hepatic macrosteatosis, which was graded on a 0–3 scale by a blinded reviewer. Kidney sections were similarly deparaffinized and stained with Periodic Acid–Schiff (PAS; Epredia, Kalamazoo, Michigan) to visualize glomerulosclerosis, which was scored from 0 to 4. A total Glomerulosclerosis Index (GSI) was then calculated. Enzyme-Linked Immunosorbent Assay (ELISA) for sFlt-1. Serum was collected from BPH/5 and C57BL/6 mice, both non-pregnant and pregnant, at the time of delivery following Ad-LacZ or Ad-hIGF administration via cardiac puncture. Blood was allowed to clot at room temperature and centrifuged at 2,000 × g for 5 min to isolate serum. Soluble Flt-1 (sFlt-1/VEGF-R1) levels were quantified using a mouse ELISA kit (R&D Systems, #MVR100, Minneapolis, Minnesota) with a sensitivity of 15.2 pg/mL. Sample Size Determination for Murine Experiments . To determine the number of BPH/5 mice required to detect a physiologically meaningful improvement in blood pressure following Ad-hIGF administration, we performed a paired-design sample-size calculation based on repeated pre- and post-injection measurements within the same mouse. Baseline systolic blood pressure in pregnant BPH/5 mice is typically 130–150 mmHg, with a within-group standard deviation of ~12 mmHg. Assuming a within-animal correlation of r = 0.7 between pre- and post-treatment measurements, the standard deviation of the paired differences is approximately 9.3 mmHg. For a biologically relevant 10 mmHg decrease in systolic blood pressure after Ad-hIGF treatment, the paired standardized effect size (Cohen’s d) is ~1.08, representing a very large within-animal effect. Using a two-sided α = 0.05, power = 80–90%, the required number of paired animals is 7. Statistical analysis. Continuous outcomes (e.g., longitudinal blood pressure and changes in blood pressure, weight, histologic scores, and morphometric measures) were analyzed separately for BPH/5 and C57BL/6 mice, when applicable, using linear mixed-effects models to compare treatment groups while accounting for correlations among repeated or clustered observations. Models included treatment as a fixed effect and incorporated additional prespecified covariates as appropriate (e.g., time and treatment-by-time interaction for longitudinal outcomes). Random intercepts were included to account for within-individual dependency (e.g., mouse-level random intercept for repeated measures; placenta- or litter-level random intercept for biological replicates). Model-based group estimates are reported as estimated marginal means with 95% confidence intervals. Binary outcomes, including live birth, fetal demise, and resorption, were analyzed using binomial regression models (logit link) with treatment as the primary predictor, fitted separately by strain when applicable. Descriptive probabilities were summarized at the litter level, and model-based mean probabilities were obtained from the fitted binomial models. Between-group comparisons were conducted based on odds ratios (ORs) with corresponding 95% confidence intervals. Multiple testing was addressed using Tukey’s significant difference adjustment for pairwise contrasts. All tests were two-sided with statistical significance assessed at α = 0.05. Modeling analyses were performed in R (version 4.5.1) using ‘lme4’ and ‘lmerTest’ for mixed-effects modeling, ‘stats’ for binomial modeling, and ‘emmeans’ for marginal means and multiplicity-adjusted contrasts. Quantitative analyses for Fig 5-9 were conducted using GraphPad Prism (v9.0 or later; GraphPad Software, San Diego, CA). Data are presented as mean ± standard deviation (SD) or median ± IQR where appropriate. For comparisons between two groups, an unpaired two-tailed Student’s t-test or Mann-Whitney U was applied where appropriate. In cases involving two independent variables (e.g., treatment and oxygen condition), a two-way ANOVA with Bonferroni correction was conducted. For the in vitro angiogenesis assay, node and mesh counts were quantified from Geltrex-coated 96-well plates using the ImageJ angiogenesis analyzer plugin. For the Ki-67 proliferation assay, flow cytometry data were analyzed using FlowJo software, with Ki-67 positivity quantified as the percentage of PE-positive cells in each treatment group. *P <0.05, **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. Exact p -values and details of statistical tests used are reported in figure legends. Discussion Intraplacental gene transfer of IGF-1 at e16 corrects preeclampsia in the BPH/5 mouse. Gene transfer occurred when symptoms of preeclampsia had already begun to manifest in BPH/5 mice. Collectively, the diastolic, systolic, and mean arterial blood pressures all normalized after IGF-1 gene transfer to the levels observed in C57BL/6 controls. BPH/5 mice treated with Ad-hIGF-1 had significantly lower urinary protein than non-pregnant BPH/5 mice and a trend toward lower urinary protein compared with BPH/5-Ad-lacZ-treated mice. The glomerulosclerosis index was also reduced by 65% compared to BPH/5 Ad-LacZ-treated mice and by 55% compared to BPH/5 surgical sham mice. Importantly, intraplacental gene transfer with Ad-hIGF-1 did not compromise fetal outcomes, with no change in the number of live births, rate of demise, or reabsorption. To validate the microvascular histologic changes observed in BPH/5 placentas, we analyzed a human placenta cohort of pregnancies complicated by early-onset preeclampsia (EOPE), finding a significant alteration in the placental villous morphology and vascularization compared to healthy and a gestational age-matched PPROM control. EOPE placentas demonstrated a reduction in microvascular density, lumens per villous, and maximum villous diameter, and an increase in total syncytial knots. The chorionic villous count did not differ between EOPE vs. PPROM. There was no difference in minimum villous diameter. The changes in placental morphometric microvascular architecture mirrored those found in BPH/5 mice. BPH/5 mice had evidence of poor vascularization and vessel architecture. Microvascular density in the BPH/5 mice was lowest in the BPH/5-Ad-lacZ-treated and BPH/5 surgical sham groups. BPH/5 mice treated with Ad-hIGF-1 and BPH/5 surgical sham mice exhibited significantly higher microvascular density, restoring levels of placental vascular integrity that became indistinguishable from those of C57BL/6 controls. Mechanistically, we found IGF-1 activated the IKKβ/NF-κB signaling axis in human microvascular placental endothelial cells, driving angiogenesis and cell proliferation. HPVECs treated with IGF-1 in the presence of pharmacologic inhibitors targeting various blocking steps within the IKKβ/NF-κB, mTOR, PI3K, and AKT pathways found that under normoxic and hypoxic conditions, IGF-1 increased tube formation, which was blocked by IKKβ/NF-κB inhibition and, to a lesser extent, mTOR inhibition. Furthermore, angiogenesis and proliferation were restored in HPVECs under serum-free normoxic and hypoxic conditions under stimulation with IGF-1. To evaluate whether IGF-1 stimulated NF-kB binding of angiogenic gene promoters’ regions through ChIP-qPCR, we found that in BeWo cells, IGF-1 promotes NF-κB p65 occupancy at key VEGFA, PlGF, and HIF-1a promoter regions. However, these findings were not seen in HPVECs. These findings suggest that the previously observed angiogenesis, proliferation, and signal-transduction effects indicate that NF-κB activity within HPVECs may act through other angiogenic gene promoter regions not assessed in these assays, and/or that cell–cell crosstalk between syncytiotrophoblasts and microvascular endothelial cells contributes to the angiogenic effects observed. It is surprising that IGF-1 gene transfer did not rescue fetal growth restriction in the BPH/5 mouse. Previously, IGF-1 placental gene transfer has been shown to correct birth weight in the MUAL model of fetal growth restriction 29 as well as in the naturally occurring FGR model in the rabbit. 28 IGF-1 has been shown not only to increase microvascular density but also to increase GLUT transporter expression and localization in both mouse models of FGR and human BeWo cells. 29 While IGF-1 corrected the preeclampsia phenotype in the BPH/5 mice, IGF-1 may fail to rescue FGR because the BPH/5 placenta may not appropriately respond to IGF-1, resulting in little or no upregulation of nutrient transporters. Even if IGF-1 impacts nutrient transporters in the BPH/5 mice, it may be that early defective placental development impacts their ability to appropriately respond. Additionally, the BPH/5 model notoriously has smaller litter sizes compared to C57BL/6 mice. This surprising lack of IGF-1 to correct FGR requires further investigation. There are additional limitations that warrant consideration. While the BPH/5 model recapitulates many features of preeclampsia, it does not fully capture the complexity of human disease. While IGF-1 activates NF-κB in trophoblasts and microvascular placental endothelial cells, alternate cellular crosstalk was not explored. Furthermore, while our findings indicate that the IGF-1 signal transduction pathway is involved in preeclampsia, we recognize that multiple phenotypes of human preeclampsia may exist. Given our prior research supporting placental gene transfer of IGF-1 corrected growth in both rabbit and mouse models of FGR, we surmise that IGF-1 is likely involved in early onset phenotypes of preeclampsia that often co-occur with FGR. 28,29,43 Additionally, our study did not find differences in sFlt-1 levels in the BPH/5 model after IGF-1 transfer. While tremendous clinical focus has centered on the downstream biomarkers like sFlt-1, this may represent outlier readouts rather than faithful indicators of mechanistic activity, especially in early-onset phenotypes. This observation is not new, as previous studies have not found that sFLt-1 was not elevated in the BPH/5 model. The placental microvascular density changes observed within our EOPE cohort were compared to those of healthy patients with differences in gestational age, which may impact vascular density. However, when compared to the PPROM patient, the results were comparable. In addition, adenovirus, while an efficient experimental means of gene transfer, is not an ideal vector for clinical application of placental gene therapy due to the inflammatory response it elicits. Clinical translation of placental gene transfer of IGF-1 will require the development of safer gene transfer platforms, such as adeno-associated virus or nanoparticles. While the results of IGF-1 intraplacental gene transfer in correcting preeclampsia in BPH/5 mice are encouraging, the implications for treating human diseases are uncertain. Further exploration of IGF-1 in preeclampsia is warranted before clinical translation can be considered. Preeclampsia continues to remain a leading cause of maternal and perinatal morbidity and mortality, with no current therapeutic options available due to an incomplete understanding of its multifactorial pathogenesis. Our observations highlight IGF-1-mediated NF-κB activation as an area of investigation and as a potential therapeutic strategy. IGF-1 restores placental angiogenesis, reduces maternal hypertension, and prevents end-organ dysfunction, while preserving maternal and neonatal outcomes in BPH/5 mice. These findings define a mechanistic link between IGF-1 signaling, endothelial function, and placental vascular health, providing a foundation for future investigation to prevent or treat severe preeclampsia. Declarations Data Availability Statement: The data have been made private due to privacy reasons in human subject research and can be made available upon reasonable request to the authors. Conflicts of interest: The authors report no conflicts of interest Funding: The authors report funding for this research from the Connecticut Children’s Connection Grant. Acknowledgements: We would like to acknowledge Dr. Jennifer Sones for her collaboration and gift of the BPH/5 line to allow this research to occur Author Contributions (CRediT) Conceptualization – KLM, SM, TMC Data Curation – KLM, SM Formal Analysis – CLK, KLM, SM Funding Acquisition – SM, TMC Investigation – AK, KLM, SM, TMC Methodology – KLM, TMC Project Administration – TMC Resources – JS, TMC Software – CLK, SM, KLM Supervision – JS, TMC Validation – KLM, SM, TMC Visualization – KLM, SM, CLK, TMC Writing – Original Draft – KLM, TMC Writing – Review & Editing – KLM, SM, CLK, JS, AK, TM References Chau, K., Welsh, M., Makris, A. & Hennessy, A. Progress in preeclampsia: the contribution of animal models. J Hum Hypertens 36 , 705-710 (2022). Gestational Hypertension and Preeclampsia: ACOG Practice Bulletin, Number 222. Obstet Gynecol 135 , e237-e260 (2020). Goldenberg, R.L., Culhane, J.F., Iams, J.D. & Romero, R. Epidemiology and causes of preterm birth. Lancet 371 , 75-84 (2008). Chappell, L.C., Cluver, C.A., Kingdom, J. & Tong, S. Pre-eclampsia. Lancet 398 , 341-354 (2021). Avorgbedor, F. , et al. Hypertension and infant outcomes: North Carolina pregnancy risks assessment monitoring system data. Pregnancy Hypertens 28 , 189-193 (2022). Henderson, J.T. , et al. Low-dose aspirin for prevention of morbidity and mortality from preeclampsia: a systematic evidence review for the U.S. Preventive Services Task Force. Ann Intern Med 160 , 695-703 (2014). Srinivas, S.K. , et al. Rethinking IUGR in preeclampsia: dependent or independent of maternal hypertension? J Perinatol 29 , 680-684 (2009). Robertson, S.A. Preventing Preeclampsia by Silencing Soluble Flt-1? N Engl J Med 380 , 1080-1082 (2019). Aubuchon, M., Schulz, L.C. & Schust, D.J. Preeclampsia: animal models for a human cure. Proc Natl Acad Sci U S A 108 , 1197-1198 (2011). Gatford, K.L., Andraweera, P.H., Roberts, C.T. & Care, A.S. Animal Models of Preeclampsia: Causes, Consequences, and Interventions. Hypertension 75 , 1363-1381 (2020). Bakrania, B.A., George, E.M. & Granger, J.P. Animal models of preeclampsia: investigating pathophysiology and therapeutic targets. Am J Obstet Gynecol 226 , S973-S987 (2022). Taylor, E.B. & George, E.M. Animal Models of Preeclampsia: Mechanistic Insights and Promising Therapeutics. Endocrinology 163 (2022). Rana, S., Burke, S.D. & Karumanchi, S.A. Imbalances in circulating angiogenic factors in the pathophysiology of preeclampsia and related disorders. Am J Obstet Gynecol 226 , S1019-S1034 (2022). Han, L., Luo, Q.Q., Peng, M.G., Zhang, Y. & Zhu, X.H. miR-483 is downregulated in pre-eclampsia via targeting insulin-like growth factor 1 (IGF1) and regulates the PI3K/Akt/mTOR pathway of endothelial progenitor cells. J Obstet Gynaecol Res 47 , 63-72 (2021). He, X. , et al. The relationship between IGF1 and the expression spectrum of miRNA in the placenta of preeclampsia patients. Ginekol Pol 90 , 596-603 (2019). Ingec, M., Gursoy, H.G., Yildiz, L., Kumtepe, Y. & Kadanali, S. Serum levels of insulin, IGF-1, and IGFBP-1 in pre-eclampsia and eclampsia. Int J Gynaecol Obstet 84 , 214-219 (2004). Umapathy, A. , et al. Molecular regulators of defective placental and cardiovascular development in fetal growth restriction. Clin Sci (Lond) 138 , 761-775 (2024). Maulik, D., Frances Evans, J. & Ragolia, L. Fetal growth restriction: pathogenic mechanisms. Clin Obstet Gynecol 49 , 219-227 (2006). Tun, W.M., Yap, C.H., Saw, S.N., James, J.L. & Clark, A.R. Differences in placental capillary shear stress in fetal growth restriction may affect endothelial cell function and vascular network formation. Sci Rep 9 , 9876 (2019). Wang, Y. & Zhao, S. in Vascular Biology of the Placenta (San Rafael (CA), 2010). Arroyo, J., Torry, R.J. & Torry, D.S. Deferential regulation of placenta growth factor (PlGF)-mediated signal transduction in human primary term trophoblast and endothelial cells. Placenta 25 , 379-386 (2004). Luo, Z.C. , et al. Maternal and fetal IGF-I and IGF-II levels, fetal growth, and gestational diabetes. J Clin Endocrinol Metab 97 , 1720-1728 (2012). Hiden, U., Glitzner, E., Hartmann, M. & Desoye, G. Insulin and the IGF system in the human placenta of normal and diabetic pregnancies. J Anat 215 , 60-68 (2009). Nawathe, A.R. , et al. Insulin-like growth factor axis in pregnancies affected by fetal growth disorders. Clin Epigenetics 8 , 11 (2016). Majumder, S., Moriarty, K.L., Lee, Y. & Crombleholme, T.M. Placental Gene Therapy for Fetal Growth Restriction and Preeclampsia: Preclinical Studies and Prospects for Clinical Application. J Clin Med 13 (2024). Su, J., Huang, X., Meng, S. & Wang, S. Investigating Serum and Placental Levels of IGF-1 and IGF-1R in Preeclampsia Patients and Their Clinical Implications. Int J Womens Health 17 , 729-738 (2025). Liu, J.P., Baker, J., Perkins, A.S., Robertson, E.J. & Efstratiadis, A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). Cell 75 , 59-72 (1993). Keswani, S.G. , et al. Intraplacental gene therapy with Ad-IGF-1 corrects naturally occurring rabbit model of intrauterine growth restriction. Hum Gene Ther 26 , 172-182 (2015). Jones, H.N., Crombleholme, T. & Habli, M. Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms. PLoS One 8 , e74632 (2013). Zhang, X. , et al. IGF-1 inhibits inflammation and accelerates angiogenesis via Ras/PI3K/IKK/NF-kappaB signaling pathways to promote wound healing. Eur J Pharm Sci , 106847 (2024). Salminen, A. & Kaarniranta, K. Insulin/IGF-1 paradox of aging: regulation via AKT/IKK/NF-kappaB signaling. Cell Signal 22 , 573-577 (2010). Liu, P. , et al. Role and mechanisms of the NF-kB signaling pathway in various developmental processes. Biomed Pharmacother 153 , 113513 (2022). Adli, M., Merkhofer, E., Cogswell, P. & Baldwin, A.S. IKKalpha and IKKbeta each function to regulate NF-kappaB activation in the TNF-induced/canonical pathway. PLoS One 5 , e9428 (2010). Rao, S., Liu, M., Iosef, C., Knutsen, C. & Alvira, C.M. Endothelial-specific loss of IKKbeta disrupts pulmonary endothelial angiogenesis and impairs postnatal lung growth. Am J Physiol Lung Cell Mol Physiol 325 , L299-L313 (2023). Iosef, C. , et al. Inhibiting NF-kappaB in the developing lung disrupts angiogenesis and alveolarization. Am J Physiol Lung Cell Mol Physiol 302 , L1023-1036 (2012). Ashida, N. , et al. IKKbeta regulates essential functions of the vascular endothelium through kinase-dependent and -independent pathways. Nat Commun 2 , 318 (2011). Dodson, R.B. , et al. Intrauterine growth restriction decreases NF-kappaB signaling in fetal pulmonary artery endothelial cells of fetal sheep. Am J Physiol Lung Cell Mol Physiol 315 , L348-L359 (2018). Davisson, R.L. , et al. Discovery of a spontaneous genetic mouse model of preeclampsia. Hypertension 39 , 337-342 (2002). Sones, J.L., Yarborough, C.C., O'Besso, V., Lemenze, A. & Douglas, N.C. Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia. PLoS One 16 , e0253453 (2021). Burton, G.J. , et al. Optimising sample collection for placental research. Placenta 35 , 9-22 (2014). Schindelin, J. , et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 9 , 676-682 (2012). Kurtz, T.W. , et al. Recommendations for blood pressure measurement in humans and experimental animals: part 2: blood pressure measurement in experimental animals: a statement for professionals from the Subcommittee of Professional and Public Education of the American Heart Association Council on High Blood Pressure Research. Arterioscler Thromb Vasc Biol 25 , e22-33 (2005). Habli, M., Jones, H., Aronow, B., Omar, K. & Crombleholme, T.M. Recapitulation of characteristics of human placental vascular insufficiency in a novel mouse model. Placenta 34 , 1150-1158 (2013). Additional Declarations There is NO Competing Interest. Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8563004","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":573503060,"identity":"415d8660-59ab-4a83-93d7-d88392f00a4e","order_by":0,"name":"Timothy Crombleholme","email":"data:image/png;base64,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","orcid":"","institution":"Connecticut Children’s Medical Center","correspondingAuthor":true,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Crombleholme","suffix":""},{"id":573503061,"identity":"933a2114-6405-45c8-b39f-2cb589561a9e","order_by":1,"name":"Kristen 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13:33:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7810175,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8563004/v1/7d665e06-b060-46d8-a877-42e7b95a0b6c.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Correction of Preeclampsia by Intraplacental Gene Transfer of IGF-1 in the BPH/5 Mouse via NF-KB Mediated Induction of Angiogenic Gene Expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePreeclampsia has a major health impact on the maternal-fetal dyad and contributes to 5-15% of maternal deaths worldwide.\u003csup\u003e1\u003c/sup\u003e Preeclampsia is associated with increased fetal and perinatal morbidity and mortality, including fetal growth restriction\u003csup\u003e2\u003c/sup\u003e, preterm birth\u003csup\u003e3\u003c/sup\u003e, and higher rates of stillbirth\u003csup\u003e4\u003c/sup\u003e and neonatal complications\u003csup\u003e5\u003c/sup\u003e. Furthermore, rates of preeclampsia in the United States have increased by 25% from 1987 to 2004\u003csup\u003e2\u003c/sup\u003e. This disorder poses a substantial economic burden, with healthcare costs estimated at 2.18 billion dollars within the first year postpartum.\u003csup\u003e2\u003c/sup\u003e Despite its considerable morbidity, mortality, and financial impact, the pathophysiology of preeclampsia is poorly understood, and preterm delivery remains the sole definitive treatment. Low-dose aspirin is recommended as a preventive therapy in high-risk pregnancies beginning at 12 weeks of gestation; however, its effectiveness is modest.\u003csup\u003e6\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eClinically, preeclampsia has been linked to maternal multi-organ failure, coagulopathic processes, and, in fetuses, can adversely affect fetal growth, leading to growth restriction.\u003csup\u003e2\u003c/sup\u003e The diagnosis in humans is established with the development of new-onset hypertension, defined as systolic blood pressure \u0026ge;140 mmHg or diastolic blood pressure \u0026ge;90 mmHg, in combination with proteinuria (\u0026gt;300 mg per 24 hours). Preeclampsia with severe features is defined by either severe-range hypertension or evidence of end-organ injury, such as elevated liver enzymes, serum creatinine \u0026gt;1.1 mg/dL, thrombocytopenia (\u0026lt;100,000/\u0026micro;L), persistent headache, visual disturbances, or pulmonary edema.\u003csup\u003e2\u003c/sup\u003e The onset of preeclampsia is typically after 20 weeks of gestation, although earlier presentations can occur and are often associated with more severe maternal and fetal outcomes. Early onset preeclampsia (EOPE) is often associated with severe fetal growth restriction (FGR) and is defined as early onset preeclampsia (EOPE) when presenting less than 34 weeks.\u003csup\u003e7\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe pathophysiology of preeclampsia is thought to be multifactorial, with key changes centering around antiangiogenic and inflammatory factors produced by the hypoxic placenta, likely secondary to aberrant spiral artery remodeling.\u003csup\u003e8,9\u003c/sup\u003e To date, animal models of preeclampsia have primarily focused on maternal treatment, with no models specifically targeting the placenta.\u003csup\u003e10-12\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have focused on diagnostic biomarker changes leading to the development of immune, vascular endothelial injury, and oxidative stress models of preeclampsia.\u003csup\u003e11\u003c/sup\u003e Prior research has suggested that the pathophysiology of preeclampsia involves dysregulated vascular tone mediated by VEGF and placental growth factor (PlGF).\u003csup\u003e13\u003c/sup\u003e These pro-angiogenic factors are antagonized in early pregnancy by excessive production of sFlt-1, a splice variant of VEGF, and later in gestation by sEng.\u003csup\u003e9\u003c/sup\u003e Although numerous molecular markers have been implicated in the pathophysiology of preeclampsia, a knowledge gap remains regarding how these factors interact to explain the overarching mechanisms driving this lethal disorder. Multiple mechanistic pathways likely contribute to disease onset, a concept supported by the more severe, early-onset forms of preeclampsia that are frequently associated with FGR. \u003csup\u003e7\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eScant research has been performed on other key known markers, like insulin-like growth factor pathway (IGF-1), and its mechanistic implications for the development of preeclampsia, especially in EOPE.\u003csup\u003e14-16\u003c/sup\u003e Endothelial function in the placenta is regulated by trophoblast-derived angiogenic mediators such as VEGF, PlGF, and IGF-1, secreted primarily by syncytiotrophoblasts and cytotrophoblasts\u003csup\u003e17\u003c/sup\u003e. Among these, IGF-1 plays a crucial role in the physiology of endothelial cells by enabling proliferation, migration, angiogenesis, and the production of vasodilator nitric oxide and VEGF.\u003csup\u003e18-25\u003c/sup\u003e IGF is notably deficient in preeclampsia and may be a more specific biomarker for diagnosing preeclampsia than sFlt-1.\u003csup\u003e26\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eMice with null mutations in IGF-1 or IGF-2 have a 60% reduction in birth weight compared to wild-type mice, and deficient IGF-1 also prevents VEGF-induced endothelial cell proliferation and survival.\u003csup\u003e27\u003c/sup\u003e We have previously demonstrated that the intraplacental gene transfer of IGF-1 corrects FGR in a naturally occurring rabbit model of fetal growth restriction\u003csup\u003e28\u003c/sup\u003e, as well as in the mesenteric uterine artery ligation model in mice.\u003csup\u003e29\u003c/sup\u003e The effects of IGF-1 in FGR may inform applications in other diseases within the context of uteroplacental insufficiency, specifically in EOPE. While these studies suggest that IGF-1 may be a promising therapeutic candidate, the precise molecular pathways through which it exerts these beneficial effects remain poorly understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIGF-1 promotes angiogenesis and inhibits inflammation through activation of the PI3K\u0026ndash;AKT\u0026ndash;IKK\u0026ndash;NF-\u0026kappa;B signaling pathway, facilitating tissue repair and wound healing.\u003csup\u003e30\u003c/sup\u003e While NF-\u0026kappa;B broadly regulates inflammation, oxidative stress, and angiogenesis in tumor cells and lymphocytes, endothelial cells exhibit unique NF-\u0026kappa;B\u0026ndash;dependent mechanisms.\u003csup\u003e31-33\u003c/sup\u003e Endothelial cell\u0026ndash;specific inhibition or deletion of IKK-\u0026beta; impairs angiogenesis during both fetal and postnatal lung development, leading to fetal growth restriction.\u003csup\u003e34-36\u003c/sup\u003e Consistent with these findings, we previously demonstrated that NF-\u0026kappa;B modulation is reduced in the pulmonary artery endothelial cells of growth-restricted fetal sheep.\u003csup\u003e37\u003c/sup\u003e Together, these studies suggest a mechanistic link between IGF-1 signaling, NF-\u0026kappa;B activation, and placental endothelial cell function.\u0026nbsp;We hypothesize that IGF-1 enhances placental endothelial proliferation and angiogenesis mediated by the IKK-\u0026beta;/NF-\u0026kappa;B signaling axis, which corrects the placental vascular dysfunction associated with preeclampsia.\u003c/p\u003e\n\u003cp\u003eTo test this hypothesis, we first examined human placentas from pregnancies with EOPE, term healthy, and gestational age-matched preterm premature rupture of membrane (PPROM) controls to determine whether microvascular density is reduced in association with endothelial dysfunction. We used a spontaneous murine model of preeclampsia, the BPH/5 mouse, to determine whether this model recapitulates features of human placental pathology. The BPH/5 line, an inbred derivative of the hypertensive BPH/2 strain originally characterized by Davisson et al., \u003csup\u003e38\u003c/sup\u003e exhibits chronic hypertension and obesity. During pregnancy, the BPH/5 mouse recapitulates key features of human preeclampsia, including impaired trophoblast invasion, inadequate spiral artery remodeling, hypertension, proteinuria, glomerular endotheliosis, and systemic endothelial dysfunction.\u003csup\u003e39\u003c/sup\u003e We compared placental microvascular density between BPH/5 and normotensive C57BL/6 controls to determine the extent to which the murine model reflects the vascular deficiencies seen in human disease. To further delineate the mechanistic relationship between IGF-1 and NF-\u0026kappa;B signaling, we performed angiogenesis and cell proliferation assays in placental endothelial cells and chromatin immunoprecipitation (ChIP) in both endothelial and BeWo cells to assess whether IGF-1 directly modulates NF-\u0026kappa;B transcriptional activity through IKK-\u0026beta; and to confirm that IGF-1-mediated angiogenesis is driven by NF-\u0026kappa;B binding to promoter regions of select angiogenic genes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFinally, to evaluate therapeutic potential, we conducted intra-placental gene transfer of human IGF-1 (hIGF-1) in pregnant BPH/5 mice and assessed maternal blood pressure, proteinuria, renal and hepatic histopathology, and placental microvascular structure. Through this integrative approach, we aimed to evaluate a causal role for impaired IGF-1/NF-\u0026kappa;B signaling in the pathogenesis of preeclampsia and to demonstrate that restoration of placental IGF-1 expression corrects endothelial dysfunction and maternal disease.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eDemographics of Human Controls and Early-Onset Preeclampsia Participants\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSix healthy participants were recruited for placental assessment, with delivery occurring at a mean gestational age of 39 weeks 4 days \u0026plusmn; 0 weeks 3 days. Among these, 4 of 6 were delivered via cesarean section and 2 of 6 via vaginal delivery. One patient with preterm premature rupture of membranes (PPROM) was recruited as a gestational age-matched control. She was diagnosed with PPROM at 30 weeks 4 days and delivered vaginally at 34 weeks 1 day.\u003c/p\u003e\n\u003cp\u003eA total of five patients with early-onset preeclampsia were diagnosed at a mean gestational age of 31 weeks 6 days \u0026plusmn; 2 weeks 2 days, with delivery at 32 weeks 5 days \u0026plusmn; 2 weeks 4 days. Two of five had pre-existing chronic hypertension. Among these patients, 2 of 5 met criteria for severe preeclampsia based on transaminitis, 2 of 5 based on elevated blood pressures, and 1 of 5 based on features of a persistent headache. Delivery mode for the EOPE cohort was 3 cesarean sections and 2 vaginal deliveries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLower Microvascular Morphometry in EOPE Placentas Compared with Healthy and PPROM Placentas\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEOPE placentas showed reduced histological CD31-positive microvascular lumens and disrupted vascular architecture compared with healthy controls, indicative of endothelial dysfunction (Fig. 1; immunofluorescence in an EOPE and early-onset fetal growth restriction [EOFGR] placenta). Morphometric analysis revealed that these microvascular alterations occurred with preservation of villous architecture. Microvascular density (MVD) was significantly lower in EOPE (Fig. 2), with a mean (95% CI) of 10.3 (6.4\u0026ndash;14.2) microvessels per high-power field, compared with healthy controls [21.4 (17.5\u0026ndash;25.3); p = 0.004] and PPROM [23.1 (14.4\u0026ndash;31.8); p = 0.036], with no difference between healthy controls and PPROM. Lumens per villus were also significantly lower in EOPE [2.8 (1.4\u0026ndash;4.1) lumens per villus] compared with healthy controls [5.3 (4.0\u0026ndash;6.5); p = 0.034], with no significant difference between EOPE and PPROM [5.5 (2.4\u0026ndash;8.6)], likely reflecting limited power. Total syncytial knot count (TSKC) was significantly higher in EOPE [11.7 (9.3\u0026ndash;14.1) knots per 20\u0026times; field] than in healthy [5.9 (3.8\u0026ndash;8.1); p = 0.007] and PPROM [4.0 (-1.3\u0026ndash;9.3); p = 0.036]. Chorionic villous counts and maximum and minimum villous diameters did not differ between groups, suggesting preserved villous architecture despite these microvascular changes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBPH/5 Placentas Also Exhibit Lower Microvascular Density, Restored by IGF-1 Gene Transfer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRepresentative immunofluorescence (IF) staining for CD31 revealed disrupted microvascular architecture in BPH/5 surgical sham controls (Fig. 3a,b) compared to C57BL/6 surgical sham placentas. BPH/5 placentas treated with Ad-IGF-1 showed marked restoration of CD31-positive microvascular structures compared to BPH/5 Ad-LacZ controls (Figure 3c\u0026ndash;f). Quantitative particle analysis confirmed that IGF-1 gene transfer significantly increased microvascular density (Fig. 3g, h), indicating improved placental vascularization in BPH/5 mice.\u003c/p\u003e\n\u003cp\u003eBPH/5 mice treated with Ad-hIGF had significantly increased MVD with the estimated marginal mean (EMM) and standard error of 18.30 \u0026plusmn; 0.87 compared to both BPH/5 Ad-LacZ (11.80 \u0026plusmn; 0.88) and BPH/5 surgical sham (11.59 \u0026plusmn; 0.97) (p \u0026lt; 0.001 for both). MVD values in BPH/5 after Ad-IGF-1 therapy were restored to levels similar to C57BL/6 surgical sham (16.65 \u0026plusmn; 1.56), which showed no significant differences compared to C57BL/6 Ad-LacZ (16.60 \u0026plusmn; 1.24) and C57BL/6 Ad-IGF-1 (19.05 \u0026plusmn; 1.27). Collectively, these findings demonstrate that placental IGF-1 gene transfer normalizes MVD in BPH/5 mice, suggesting restoration of placental vascular integrity in the preeclamptic phenotype (Fig. 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF-1 Restores Angiogenesis and Proliferation in Human Placental Microvascular Endothelial Cells Under Normoxia and Hypoxia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine whether IGF-1 enhances endothelial function in the human placenta, we assessed angiogenic and proliferative responses in human placental microvascular endothelial cells (HPVECs) in normoxia (21% O₂) and hypoxia (5% O₂). In normoxia, IGF-1 treatment significantly increased tube formation, as reflected by a marked rise in angiogenic node formation compared with untreated controls. The extent of network restoration was comparable to the positive control, indicating that IGF-1 robustly promotes angiogenesis under normoxic conditions (Fig. 5A).\u003c/p\u003e\n\u003cp\u003eIn physiologic hypoxic placental conditions (5% FiO2), IGF-1 supplementation restored angiogenesis to levels equivalent to those observed in the positive control, demonstrating that IGF-1 effectively rescues endothelial function in conditions mimicking intrauterine hypoxia (Fig. 5B). Consistent with these findings, Ki-67 immunostaining revealed that IGF-1 significantly increased the proportion of proliferating HPVECs under both normoxia and hypoxia (Fig. 5C, D). Under each condition, the proliferative index in IGF-1\u0026ndash;treated cells matched that of the positive control, confirming IGF-1\u0026rsquo;s potent proliferative effect on placental microvascular endothelium. Collectively, these results demonstrate that IGF-1 enhances both angiogenesis and proliferation in HPVECs in both normoxic and hypoxic conditions, supporting a role for IGF-1 in maintaining placental vascular integrity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF-1\u0026ndash;Induced Angiogenesis and Proliferation Depend Primarily on Ikk\u0026beta;/NF-\u003c/strong\u003e\u003cstrong\u003ek\u003c/strong\u003e\u003cstrong\u003eB Signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo delineate the signaling mechanisms underlying IGF-1\u0026ndash;mediated endothelial activation, HPVECs were treated with IGF-1 in the presence of pharmacologic inhibitors targeting IKK\u0026beta;/NF-\u0026kappa;B (BAY-11-7082), mTOR (rapamycin), PI3K (LY-294002), or Akt (GDC-0068). Under normoxic conditions, IGF-1 significantly enhanced tube formation, an effect abolished by IKK\u0026beta; inhibition and partially reduced by rapamycin, suggesting contributions from both NF-\u0026kappa;B and mTOR pathways. In contrast, PI3K and Akt blockade had minimal impact (Fig. 6A). Under hypoxia, IGF-1 induced a robust angiogenic response that was completely abolished by IKK\u0026beta; inhibition and significantly reduced by rapamycin, while PI3K and Akt inhibitors modestly decreased tube formation. IGF-1 increased HPVEC proliferation under normoxia, which was significantly attenuated by IKK\u0026beta; and, to a lesser extent, mTOR inhibition, but not by inhibitors of PI3K or Akt. Similarly, IGF-1\u0026ndash;driven proliferation under hypoxia was markedly reduced by IKK\u0026beta; inhibition and blunted by rapamycin. These findings identify IKK\u0026beta;/NF-\u0026kappa;B as an important mediator of IGF-1\u0026ndash;induced angiogenic and proliferative responses in placental endothelial cells, with mTOR contributing as a secondary pathway. Together, they highlight NF-\u0026kappa;B activation as an important signal transduction pathway by which IGF-1 preserves placental vascular function under stress (Fig. 6)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF-1 Enhances NF-\u0026kappa;B Recruitment to Angiogenic Gene Promoters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test whether IGF-1 directly promotes NF-\u0026kappa;B\u0026ndash;dependent transcription of angiogenic genes, we performed chromatin immunoprecipitation followed by qPCR (ChIP\u0026ndash;qPCR) using an antibody against phospho-NF-\u0026kappa;B p65 in both BeWo and HPVECs. Promoter occupancy was analyzed at promoter regions for vascular endothelial growth factor A (\u003cem\u003eVEGFA)\u003c/em\u003e, \u0026nbsp;placental growth factor (PlGF), and hypoxia inducible factor 1 alpha (\u003cem\u003eHIF1a)\u003c/em\u003e genes in trophoblasts and placental microvascular endothelial cells under basal, lipopolysaccharide (LPS)-stimulated, and IGF-1\u0026ndash;stimulated conditions.\u003c/p\u003e\n\u003cp\u003eIn BeWo cells, IGF-1 stimulation led to a pronounced enrichment of p65 at the \u003cem\u003eVEGFA\u003c/em\u003e gene (~15-fold increase relative to control), whereas LPS produced only moderate (~5-fold) enrichment (Fig. 7A). At the \u003cem\u003ePGF\u003c/em\u003e gene, IGF-1 induced a ~60-fold enrichment compared to ~5-fold for LPS (Fig. 7B). Similarly, p65 binding to the \u003cem\u003eHIF1A\u003c/em\u003e increased ~25-fold following IGF-1 stimulation, in contrast to ~3-fold with LPS (Fig. 7C). In HPVECs, IGF-1 stimulation showed no difference in VEGFA, PlGF or HIF1A compared to unstimulated control (Fig. 8).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese results suggest that IGF-1 selectively enhances NF-\u0026kappa;B p65 recruitment to promoters of VEGF, PlGF, HIF-1a\u0026nbsp;in BeWo cells but not HPVECs, distinguishing IGF-1\u0026ndash;driven NF-\u0026kappa;B activation from the classical inflammatory NF-\u0026kappa;B response induced by LPS. This finding suggests a mechanistic link between IGF-1 signaling and transcriptional activation of placental angiogenesis pathways. The attenuation of angiogenesis in HPVECs with Bay-11-7082 compound suggests that NF-kB stimulates angiogenic gene expression, however the lack of binding to promoters of VEGFA,\u0026nbsp;PlGF\u0026nbsp;and HIF-1a suggests that other angiogenic genes may be responsible in HPVECs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF-1 Gene Transfer Normalized Systolic, Diastolic, And Mean Arterial Blood Pressures in the BPH/5 Mice Compared to C57BL/6 Controls\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy groups:\u003c/strong\u003e C57BL/6 Ad-LacZ treated (n=5), C57BL/6 Ad-IGF-1 treated (n=8), BPH/5 Ad-LacZ treated (n=5), and BPH/5 Ad-IGF-1 treated (n=6)\u0026mdash;with observations recorded per animal across the different physiological time points (non-pregnant, 1st trimester (e5-e13), pre-injection (e14-e16), and post-treatment (e19-e21), as reflected in the summary data as follows.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSystolic Blood Pressure (SBP): SBP was significantly higher in BPH/5 mice compared with C57BL/6 mice post-treatment with Ad-LacZ (157.5 \u0026plusmn; 7.7 vs. 120.2 \u0026plusmn; 7.7 mmHg; p = 0.0013) (Fig. 9a). Furthermore, injection with Ad-IGF-1 showed a lower SBP (124.3 mmHg) compared to injection with Ad-LacZ (157.5 mmHg) (p\u0026lt;0.0001). Linear mixed-effects modeling revealed a significant treatment effect in BPH/5 mice. Compared with Ad-LacZ, post-injection changes with Ad-IGF-1 were not statistically significant (p \u0026gt; 0.05), whereas significant post-injection changes were observed following Ad-LacZ, with notable differences in changes between groups. In contrast, no significant differences in changes from any reference period to post-injection between Ad-IGF-1 and Ad-LacZ were observed in C57BL/6 mice (all p \u0026gt; 0.24) (Fig. 10a,b). Thus, Ad-IGF-1 therapy significantly attenuates systolic blood pressure in BPH/5 mice, resulting in lower post-injection SBP trajectories compared with the hypertensive response observed following Ad-LacZ treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiastolic Blood Pressure (DBP):\u003c/strong\u003e The mean DBP post-injection for BPH/5 Ad-LacZ mice was 123.2 mmHg compared to 92.0 mmHg in BPH/5 mice with Ad-IGF-1 (p=0.0158) (Fig. 9b). Compared to BPH/5 mice treated with Ad-LacZ, those treated with Ad-IGF-1 showed no significant post-injection changes across any reference period (p \u0026gt; 0.05). In contrast, Ad-LacZ-treated mice showed increased DBP consistently across reference periods, with a significant difference in the change from first trimester to post-injection between groups (post-injection \u0026ndash; first trimester -4.33 in IGF vs. 35.1 in Ad-LacZ, p=0.025). In C57BL/6 mice, no significant differences in changes from any reference period to post-injection were observed between Ad-IGF-1 and Ad-LacZ (Fig. 10c,d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMean Arterial Pressure (MAP):\u0026nbsp;\u003c/strong\u003eIGF treatment significantly lowered the MAP in BPH/5 mice compared to those treated with Ad-LacZ (p = 0.0026), normalizing it to levels comparable with C57BL/6-Ad-IGF-1 treated mice (p = 0.786) (Fig. 9C). Similarly, in BPH/5 mice, those treated with Ad-LacZ and Ad-IGF-1 showed significant differences in the non-pregnant to post-injection change (post-injection \u0026ndash; non-pregnant -4.48 in Ad-IGF-1 vs. 26.54 in Ad-LacZ) and the first trimester to post-injection change (post-injection \u0026ndash; first trimester 5.53 in Ad-IGF-1 vs. 36.80 in Ad-LacZ). In C57BL/6 mice, no significant differences between Ad-IGF-1 and Ad-LacZ were detected in changes from the non-pregnant or first-trimester periods to post-injection (p = 0.725 and p = 0.887, respectively), whereas a modest difference was observed for the post \u0026ndash; pre-injection 12.19 in Ad-IGF-1 vs. -20.95 in Ad-LacZ (p = 0.044) (Fig. 10e,f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these findings indicate that Ad-IGF-1 treatment therapy improved systolic, diastolic, and mean arterial pressures following injection in BPH/5 mice compared with Ad-LacZ\u0026ndash;treated controls. When blood pressure was analyzed longitudinally using post-treatment\u0026ndash;referenced deltas, systolic hypertension emerged as the most affected parameter, remaining consistently elevated in the Ad-LacZ cohort across all comparisons, whereas effects on diastolic pressure and mean arterial pressure were more modest and less consistently statistically significant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine Proteinuria Is Reduced by Ad-hIGF Treatment In BPH/5 Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUrinary protein was tracked across time at non-pregnant, first-trimester, pre-, and post-injection time points. Among the BPH/5 cohorts, estimated marginal means (EMMs) \u0026plusmn; standard errors (SEs) were calculated for each strain with Ad-IGF-1 or Ad-LacZ using a linear mixed-effect model (Fig. 11). In the BPH/5 cohort, Ad-IGF-1 treatment reduced the post-injection change in urinary protein in comparison to the non-pregnant state (\u0026minus;0.44 \u0026plusmn; 0.17) compared with Ad-LacZ (0.09 \u0026plusmn; 0.16), with a significant change difference (\u0026Delta;EMM = \u0026minus;0.53 \u0026plusmn; 0.23; p = 0.039). No significant trends were seen for post-first trimester or post-pre-injection comparisons.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLitter Size, Rate of Demise, Reabsorption, And Pup Weight Were Not Altered by Therapy With IGF-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAd-IGF-1 therapy does not affect live birth rates in BPH/5 mice (Figure 12a). The probability of live birth for BPH/5 Ad-IGF-1 was 0.29 (0.18-0.42), BPH/5 Ad-LacZ was 0.47 (0.34-0.61), and BPH/5 surgical sham was 0.50 (0.34-0.66), and pairwise comparisons showed no difference in live birth rate between groups.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIGF-1 treatment does not affect the rate of pup demise in BPH/5 mice (Figure 12b), with\u003c/p\u003e\n\u003cp\u003ethe number of demises per litter being low across all experimental groups. In C57BL/6, surgical sham showed higher odds of demise than Ad-LacZ (OR 5.82, p \u0026lt; 0.001) and Ad-IGF-1 (OR 3.35, p = 0.032), whereas no significant difference was observed between Ad-LacZ and Ad-IGF-1 (p = 0.505). \u003c/p\u003e\n\u003cp\u003eThe number of resorptions per litter was generally low across all groups (Figure 12c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn BPH/5 mice, Ad-IGF-1-treated mice showed a higher probability of resorption than Ad-LacZ-treated mice (OR 10, p = 0.002), while the surgical sham group showed low resorption. In C57BL/6 mice, resorption rates were generally low, with higher odds in Ad-LacZ\u0026ndash;treated mice compared with Ad-IGF-1-treated mice (OR = 12.5, p = 0.042). However, because the direction of the effect is opposite between strains, this trend is likely unreliable and requires further investigation.\u003c/p\u003e\n\u003cp\u003eIGF-1 therapy did not improve pup weight in BPH/5 mice (Fig. 12d). No differences in pup weight were observed between Ad-IGF-1 and Ad-LacZ groups within either strain, whereas surgical sham pups weighed more than those from virally treated pregnancies. These differences may reflect physiological responses associated with targeted intraplacental delivery, independent of vector or transgene identity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF treatment reduces glomerulosclerosis in BPH/5 mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe glomerulosclerosis index (GSI) was markedly elevated in BPH/5 mice compared with C57BL/6 controls, consistent with the increased renal pathology observed in the BPH/5 mice. Gene transfer with Ad-LacZ did not significantly alter GSI compared with the BPH/5 surgical sham, confirming it as a suitable control. The estimated marginal mean and standard error (EMM \u0026plusmn; SE) glomerulosclerosis index (GSI) for BPH/5 Ad-LacZ mice was 232.20 \u0026plusmn; 27.17, compared with 98.20 \u0026plusmn; 21.48 in BPH/5 mice treated with Ad-IGF-1 (p=0.006), representing an approximately 58% reduction with IGF-1 treatment relative to those treated with Ad-LacZ and 47.2% to BPH/5 surgical shams. Consistent with these quantitative findings, Figs. 13a and 13b show clear evidence of improved glomerular architecture and reduced glomerulosclerosis following Ad-IGF-1 treatment. Note, the GSI in BPH/5 IGF-treated mice was not significantly different from C57BL/6-Ad-IGF-1 controls, indicating that IGF-1 restored glomerular histology toward baseline levels.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLiver dysfunction does not accompany the BPH/5 model of pre\u003c/strong\u003e\u003cstrong\u003e‑\u003c/strong\u003e\u003cstrong\u003eeclampsia\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver macrosteatosis was assessed in C57BL/6 and BPH/5 mice under non-pregnant (NP), Ad-LacZ-treated, Ad-IGF-1-treated, and surgical sham conditions. Livers were graded from 0-3, with 0 representing no evidence of macrosteatosis and 3 representing global macrosteatosis. BPH/5 mice had higher levels of macrosteatosis in the non-pregnant state, estimated marginal mean and standard error (EMM \u0026plusmn; SE) (2.40 \u0026plusmn; 0.36) compared to BPH/5 surgical sham (0.75 \u0026plusmn; 0.40), BPH/5 Ad-LacZ (0.70 \u0026plusmn; 0.36), and BPH/5 Ad-IGF-1 (0.64 \u0026plusmn; 0.31), p \u0026le; 0.034. No significant differences were observed among the treated or sham groups (all p \u0026gt; 0.99). These results indicate strain-specific elevation in liver measurements in BPH/5 mice, with no detectable effect of IGF-1 treatment (Fig. 14).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIGF treatment does not alter Soluble Fms-like Tyrosine Kinase-1 (sFlt-1)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003esFlt-1 levels were measured in C57BL/6 and BPH/5 mice under baseline non-pregnant (NP) conditions and following Ad-LacZ or Ad-IGF-1 treatment. In C57BL/6 mice, baseline estimated marginal means and standard error for s-Flt were low (6,183 \u0026plusmn; 1,943 pg/ml, n=6 mice) and increased in pregnancy as expected, but with no significant difference in sFlt-1 between treatments with Ad-LacZ (22,558 \u0026plusmn; 2054, n=6) and Ad-IGF-1 (25,327 \u0026plusmn; 4,095 pg/ml, n=7). In BPH/5 mice, baseline sFlt-1 was very low (3,379 \u0026plusmn; 3896 pg/ml, n = 4) and rose in pregnancy with Ad-LacZ (25,755 \u0026plusmn; 4498, n = 3) and Ad-IGF-1 treatment (26,090 \u0026plusmn; 2945 pg/ml, n = 7). The difference between these treatments was minimal and not statistically significant (p = 0.998). Overall, IGF did not further alter sFlt-1 compared to controls within either strain.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eHuman Placental Collection.\u0026nbsp;\u003c/strong\u003eA prospective study enrolled patients diagnosed with EOPE, term healthy controls, and PPROM for placental collection from April 2024 to August 2025 following Institutional Review Board (IRB) approval from UConn Health (IRB# 24-099J-1). Informed consent was obtained from all patients. Placentas were harvested according to prior guidelines set out by Burton et al.\u003csup\u003e40\u003c/sup\u003e A placental biopsy, approximately 3 cm x 3 cm, was taken in a random lateral quadrant free from infarctions, with care to avoid the umbilical cord insertion. The placenta sample was further dissected, removing the decidual and chorionic plates to isolate the mid-placental surface for analysis. Samples were fixed in 10% neutral-buffered formalin for 24 hours and stored in 70% ethanol until paraffin embedding. The tissues were cryosectioned at 5 \u0026micro;m thickness and mounted onto Superfrost Plus glass slides for later staining.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrovascular Density Assessment in Human Placentas.\u0026nbsp;\u003c/strong\u003eSamples were stained for CD31 using the above immunofluorescence protocol with primary antibody for CD31 at 1:100 (Thermo Fisher, Waltham, MA, USA), followed by Goat Anti-Rabbit IgG (H+L) Alexa Fluor 488 Conjugated Secondary Antibody (Thermo Fisher) at 1:200. Blinded quantitative morphometric analysis was performed on hematoxylin and eosin\u0026ndash;stained placental sections. Two sections per placenta were analyzed. Chorionic villus count (CVC) and maximal and minimal chorionic villus diameter were measured at 10\u0026times; magnification in 5 randomly selected fields per section.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal syncytial knot count (TSKC) was assessed at 20\u0026times; magnification in 5 randomly selected fields. Luminal count (LC) per villus and total microvascular density were quantified at 40\u0026times; magnification in oil in 10 randomly selected regions per section. All measurements were performed in a blinded manner using digital image analysis software to ensure reproducibility and minimize observer bias. Samples were imaged using a Zeiss fluorescent microscope (Oberkochen, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence.\u0026nbsp;\u003c/strong\u003eFrozen tissue samples embedded in OCT compound were sectioned coronally to visualize all layers, including the maternal decidua, the junctional zone with spongiotrophoblasts and glycogen cells, the labyrinth zone where maternal and fetal blood spaces intermingle, and the fetal-facing chorionic plate containing fetal vessels. The tissues were cryosectioned at 5 \u0026micro;m thickness and mounted onto Superfrost Plus glass slides. Frozen sections stored at -80\u0026deg;C were warmed to room temperature and placed in (50%-100%) ethanol for 5 minutes at room temperature. Slides were then placed in a 1:1 methanol/acetone mixture at -20\u0026deg;C for 10 minutes. Slides were permeabilization and blocked for intracellular antigen detection for 1 hour using 30 ml PBS, 60 \u0026mu;l 0.1% Triton X-100, 60 \u0026mu;l Tween, 60 \u0026mu;l Triton. After blocking, slides were incubated overnight using a 1:100 dilution of Anti-mouse/rCD31 goat (Invitrogen, Waltham, MA, USA).). The next day, samples were washed with PBS and incubated for 1 hour in a 1:200 dilution of Alex 546 Donkey anti-Goat (Invitrogen). Tissue sections were incubated with DAPI (1 \u0026micro;g/mL in PBS) for 10 minutes at room temperature to stain nuclei, followed by a final PBS wash. Imaging - Samples were imaged using a Zeiss fluorescence microscope with filter sets appropriate for DAPI (excitation 358 nm, emission 461 nm) and Alexa Fluor 546 (excitation 556 nm, emission 573 nm).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine Placental Microvascular Density Assessment.\u0026nbsp;\u003c/strong\u003eFetal endothelial microvascular density (MVD, expressed as % area or normalized vessel coverage) was quantified in BPH/5 and C57BL/6 placentas, including surgical sham controls, Ad-LacZ, and Ad-hIGF treated groups, following immunohistochemical staining for CD31, an endothelial cell marker of placental vasculature. Images were acquired at 10\u0026times; magnification in three to four representative regions per placenta using a Zeiss fluorescent microscope. Placental morphometric analysis was performed in ImageJ (NIH, Bethesda, MD, USA).\u003csup\u003e41\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003eFor image processing, red-channel images were converted to 8-bit, binarized, and holes were filled. The scale was uniformly set across all 10\u0026times; images (distance in pixels = 1; known distance = 83.82; unit = \u0026micro;m). Particle analysis was performed with a size threshold of 25\u0026ndash;\u0026micro;m\u0026sup2; and circularity set to 1.0, following manual thresholding. Extracted parameters included vessel count, total vessel area, average vessel size, and % area. The tissue area of each 10\u0026times; image was 1040 \u0026times; 1388 pixels (1.013 \u0026times; 10\u0026sup1;⁰ \u0026micro;m\u0026sup2;). Microvascular density was calculated as total vessel area (\u0026micro;m\u0026sup2;) divided by total tissue area (\u0026micro;m\u0026sup2;).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman Placental Microvascular Endothelial Cell (HPVEC) Culture.\u0026nbsp;\u003c/strong\u003eHuman Placental Microvascular Endothelial Cells (HPVECs) were obtained from ScienceCell Laboratories (Carlsbad, CA, USA) and maintained in the manufacturer-recommended endothelial cell medium supplemented with growth factors and 5% CO₂\u0026nbsp;at 37\u0026deg;C under normoxia (21% O₂). Culture plates were pre-coated with fibronectin to promote cell adhesion. The culture medium was replaced every two days, and cells were passaged upon reaching 80\u0026ndash;90% confluency. All experiments were performed using cells between passages 3 and 5 and in replicates (n \u0026ge; 6).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eReagents for Human In Vitro Studies and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eChromatin Immunoprecipitation Assay\u003c/strong\u003e\u003cstrong\u003e.\u0026nbsp;\u003c/strong\u003eIGF-1 LR3 was sourced from PeproTech (Rocky Hill, NJ, USA). Pharmacologic inhibitors\u0026mdash;such as BAY-11-7082 (IKK-\u0026beta; inhibitor), Rapamycin (mTOR inhibitor), GDC-0068 (pan-AKT inhibitor), and LY-294002 (pan-PI3K inhibitor)\u0026mdash;came from MedChemExpress (Monmouth Junction, NJ, USA). The Ki67-PE antibody for flow cytometry was obtained from BD Biosciences (San Jose, CA, USA). Phospho-NF-\u0026kappa;B p65 and the SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit for chromatin immunoprecipitation assay were procured from Cell Signaling Technology (Danvers, MA). For RNA isolation, cDNA synthesis, and RTqPCR analyses, the RNeasy Mini Kit was purchased from Qiagen (Germantown, MD, USA), and the iScript\u0026trade; gDNA Clear cDNA Synthesis Kit, iTaq\u0026trade; Universal SYBR\u0026reg; Green Supermix, were obtained from Bio-Rad (Hercules, CA, USA). All primers, including mouse \u003cem\u003eIgf1\u003c/em\u003e and human \u003cem\u003eVegfa\u003c/em\u003e, \u003cem\u003ePgf\u003c/em\u003e, and \u003cem\u003eHif1a,\u003c/em\u003e were obtained from Bio-Rad (Hercules, CA, USA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Angiogenesis Assay.\u0026nbsp;\u003c/strong\u003eHuman placental microvascular endothelial cells (HPVECs; ScienceCell) were seeded on Geltrex\u0026trade; basement membrane matrix (Thermo Fisher Scientific) to assess angiogenesis. Ninety-six\u0026ndash;well plates were coated with 50 \u0026micro;L of Geltrex and incubated at 37 \u0026deg;C for 30 min to allow solidification. Cells (2.5 \u0026times; 10⁴ per well) were cultured in complete or incomplete endothelial medium supplemented with recombinant human IGF-1 (100, 250 or 500 ng mL⁻\u0026sup1;). IGF-1 at 250 and 500 ng mL⁻\u0026sup1; enhanced tube formation; 500 ng mL⁻\u0026sup1; was used for subsequent assays.\u003c/p\u003e\n\u003cp\u003eTo evaluate pathway involvement, cells were then co-treated with IGF-1 (500 ng mL⁻\u0026sup1;) and one of four pharmacologic inhibitors\u0026mdash;BAY-11-7082, rapamycin, GDC-0068, or LY-294002\u0026mdash;at their IC₅₀\u0026nbsp;concentrations. Tube formation was monitored for 18 hours under normoxia (21% O₂) or hypoxia (5% O₂) using a controlled hypoxia chamber. Images were acquired on an inverted phase-contrast microscope, and tube networks were quantified using the Angiogenesis Analyzer plugin in ImageJ.\u003csup\u003e41\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Vitro Proliferation Assay.\u0026nbsp;\u003c/strong\u003eCell proliferation was quantified by Ki-67 immunostaining and flow cytometry. HPVECs (2.5 \u0026times; 10⁴ per well) were seeded in 24-well plates and maintained overnight in low-serum medium (2% FBS) to promote survival and synchronize cells in a quiescent state. The following day, cells were treated with complete medium (10% FBS), low-serum medium (2% FBS), IGF-1 (500 ng mL⁻\u0026sup1;), or IGF-1 (500 ng mL⁻\u0026sup1;) combined with one of the inhibitors (BAY-11-7082, rapamycin, GDC-0068, or LY-294002) at their IC₅₀\u0026nbsp;concentrations in low serum medium. Cultures were maintained for 48 h with medium replacement at 24 h under normoxic (21% O₂) or hypoxic (5% O₂) conditions.\u003c/p\u003e\n\u003cp\u003eAfter treatment, cells were trypsinized, fixed by dropwise addition of ice-cold 70% ethanol, and stored at \u0026minus;20 \u0026deg;C for \u0026ge; 2 h. Fixed cells were washed twice with BD staining buffer, resuspended in 100 \u0026micro;L buffer, and incubated with 10 \u0026micro;L PE-labelled anti\u0026ndash;Ki-67 antibody (BD Biosciences) for 30 min at room temperature in the dark. After washing, samples were analyzed by flow cytometry. All experiments were performed in biological sextuplicates (n = 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromatin immunoprecipitation and quantitative PCR (ChIP\u0026ndash;qPCR) in BeWo and HPVECs.\u0026nbsp;\u003c/strong\u003eChromatin immunoprecipitation was performed using the SimpleChIP\u0026reg; Enzymatic Chromatin IP Kit (magnetic beads; Cell Signaling Technology, Danvers, MA, USA)) according to the manufacturer\u0026rsquo;s protocol. BeWo as well as HPVECs were cross-linked with 1% formaldehyde for 10 min at room temperature, and the reaction was quenched with 125 mM glycine. Cells were washed with ice-cold PBS, collected, and lysed to isolate nuclei. Chromatin was digested with micrococcal nuclease and briefly sonicated to yield DNA fragments of 150\u0026ndash;900 bp.\u003c/p\u003e\n\u003cp\u003eImmunoprecipitation was carried out overnight at 4 \u0026deg;C using an antibody against phospho\u0026ndash;NF-\u0026kappa;B p65 (Cell Signaling Technology, Cat. #71254) or normal rabbit IgG (negative control). Immune complexes were captured with magnetic beads, washed sequentially, and eluted. Cross-links were reversed by incubation at 65 \u0026deg;C, and DNA was purified using spin columns provided in the kit.\u003c/p\u003e\n\u003cp\u003ePurified DNA was analyzed by quantitative PCR using SYBR Green master mix (Bio-Rad, Hercules, CA, USA) with primer sets for VEGFA, PlGF, and Hif-1A (Bio-Rad). Fold enrichment was calculated as the percent input normalized to IgG controls. Reactions were performed in technical duplicates from three independent biological replicates.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e. Virgin control C57BL/6 mice at 8-12 weeks of age were obtained from prior in-house colonies. Virgin BPH/5 mice were originally gifted from Dr. Jennifer Sones (Colorado State University) and maintained as an in-house colony. C57BL/6 \u0026nbsp;mice were chosen as controls, as they have been used in previous BPH/5 studies and were used in the initial eight-way cross to derive the final BPH-5 strain, and are one of the most original relatives to the BPH/5 line.\u003csup\u003e38\u003c/sup\u003e The University of Connecticut Animal Care and Use Committee approved all experiments. Care of the mice met all standards set forth by the National Institute of Health (NIH) guidelines on the care and use of animals, regulations set forth by the United States Drug Association (USDA), and the Animal Veterinary Medical Association Panel Euthanasia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdenoviral Constructs\u003c/strong\u003e. The Ad-hIGF-1 adenoviral construct is a replication-defective Ad5 vector (E1/E3 deleted) under the control of the CMV promoter. Human IGF (h-IGF)-1 construct was generously supplied by L. Sweeney (University of Pennsylvania, Philadelphia, PA). The recombinant adenovirus carrying the \u0026beta;-galactosidase transgene (Ad-LacZ) was obtained from J. Wilson (University of Pennsylvania) and was also an Ad5 vector (E1/E3 deleted) under a CMV promoter. The parental adenovirus for Ad-hIGF-1 was Ad-Easy, and for Ad-LacZ was \u003cem\u003edl\u003c/em\u003e7001. Viruses were prepared with 293 cells and purified and stored in 50% glycerol at -80C and viral titers were determined using absorbance at 260 nm and diluted 1:10 with phosphate-buffered saline (PBS) before use in the mice to prevent glycerol toxicity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTimed Mating.\u0026nbsp;\u003c/strong\u003eAll animals were housed in a facility with constant temperature and humidity and under a controlled 12:12 h light/ dark cycle and allowed free access to standard chow and water. All mice (C57BL/6 and BPH/5) were time mated (based on Charles River breeding protocol: day 1 of gestation is defined as the observed day of vaginal plug after male and female mating).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlood Pressure Monitoring\u003c/strong\u003e. BPH/5 mice have significantly elevated baseline MAP compared with control mice before pregnancy (128\u0026plusmn;5 versus 106\u0026plusmn;7 mm Hg, respectively; P\u0026lt;0.01).\u003csup\u003e38\u003c/sup\u003e MAP remains stable in BPH/5 and C57BL/6 mice throughout the first 2 weeks of pregnancy.\u003csup\u003e38\u003c/sup\u003e However, beginning in the last trimester (day 14), MAP begins to rise even further in BPH/5 mice, increasing to peak levels just before delivery. MAP returns to pre-pregnancy levels by postpartum day 2 or 3. In sharp contrast, MAP falls at the end of the second trimester in C57BL/6 mice for a short period but returns to pre-pregnancy levels several days before delivery, where it remains throughout the postpartum period.\u003csup\u003e38\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC57BL/6 and BPH/5 mice were acclimated to blood pressure tail cuff monitoring before breeding. Blood pressure monitoring occurs as follows. All experimental mice were habituated to the CODA 8 non-invasive blood pressure system pre-pregnancy to allow habituation of tail cuff monitoring as described in prior habituation protocols.\u003csup\u003e42\u003c/sup\u003e Murine tail cuff monitoring was performed with the CODA 8 non-invasive blood pressure acquisition system (Kent Scientific, Torrington, CT), which uses VPR to detect blood pressure based on volume changes in the tail. The CODA system was set and calibrated with factory standards. Patency of the occlusion and VPR cuffs was checked routinely. The blood pressure measurements were conducted in a designated quiet area, and the CODA incubator was preheated to 37˚C to maintain body temperature during each measurement. Mice were encouraged to walk into the restraint tubes, and the tube end holders were adjusted to prevent excessive movement. The test clamp was placed at the end of each mouse tail, and the blood pressure was measured after the heartbeat was noted to have stabilized. To measure blood pressure, the occlusion cuff is inflated to 250 mm Hg and deflated over 20 s. The VPR sensor cuff detects changes in the tail volume as the blood returns to the tail during the occlusion cuff deflation. The minimum volume change was set as 15 \u0026mu;L. Each recording session consisted of 15 to 25 inflation and deflation cycles per set, of which the first 5 cycles were \u0026ldquo;acclimation\u0026rdquo; cycles and were not used in the analysis, whereas the following cycles were used. When possible, three measurements were recorded, and the average measurement was reported for that evaluation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStudy Design for Animal Experimentation\u003c/strong\u003e. For experiments, C57BL/6 and BPH/5 mice were time-mated, and blood pressure was recorded pre-pregnancy, in the first trimester (e5-e13), pre-injection (e14-e16), and post-injection at the time of harvest (e19-e21). Urine was collected at the same time points. To test the hypothesis that IGF-1 can treat the preeclampsia phenotype, a laparotomy was performed on e16, and both uterine horns were exposed. 1x10\u003csup\u003e8\u003c/sup\u003e plaque-forming units (pfu) of Ad-hIGF-1 or Ad-LacZ was administered to each placenta along with the placement of a permanent cerclage with silk. Final delivery was performed at embryonic day 21 (E21) via laparotomy, with collection of pups, placentas, and maternal liver and kidney tissues. Surgical sham BPH/5 and C57BL/6 mice underwent laparotomy on e16 with cerclage placement, with final harvest similarly at e21. At the time of harvest, fetal pup weights, live births, and the number of resorptions were noted. Placentas were frozen in OCT media for future cryo-sectioning and immunohistochemical analysis. Maternal liver and kidney were collected and paraffin-embedded. A cardiac needle aspiration was again performed for serological analysis of sFlt-1. Primary outcome measures were improvement in the preeclampsia phenotype through reduction in blood pressure and proteinuria. Secondary outcome data were fetal pup weight, litter size, reabsorption rate, sFLT-1 levels, glomerulosclerosis, hepatic macrosteatosis, and placental microvascular density of fetal vascular endothelial cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMurine Urine Protein Assessment\u003c/strong\u003e. Mice were restrained by gently grasping the base of the tail with the dominant hand and placing the thumb on either side of the neck with the non-dominant hand. The tail was then released while gently stroking the belly, and the animal was held over the collection device. We used 0.5-ml Eppendorf tubes to collect urine. Urine was frozen at \u0026minus;80\u0026deg;C until analysis for total protein content using the protein assay kit (Bio-Rad) according to the manufacturer\u0026rsquo;s instructions, which required 5 microliters of sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e. Murine Liver and kidney tissues were fixed in 10% neutral-buffered formalin for 24 hours and stored in 70% ethanol until paraffin embedding. Paraffin blocks were sectioned at 5 \u0026micro;m and mounted on Superfrost Plus slides, which were baked to ensure adherence. Slides were deparaffinized using Histo-Clear followed by a graded ethanol series (100%\u0026ndash;70%) and rinsed in deionized water. Liver sections were stained with hematoxylin and eosin (H\u0026amp;E) to evaluate hepatic macrosteatosis, which was graded on a 0\u0026ndash;3 scale by a blinded reviewer. Kidney sections were similarly deparaffinized and stained with Periodic Acid\u0026ndash;Schiff (PAS; Epredia,\u0026nbsp;Kalamazoo, Michigan) to visualize glomerulosclerosis, which was scored from 0 to 4. A total Glomerulosclerosis Index (GSI) was then calculated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-Linked Immunosorbent Assay (ELISA) for sFlt-1.\u0026nbsp;\u003c/strong\u003eSerum was collected from BPH/5 and C57BL/6 mice, both non-pregnant and pregnant, at the time of delivery following Ad-LacZ or Ad-hIGF administration via cardiac puncture. Blood was allowed to clot at room temperature and centrifuged at 2,000 \u0026times; g for 5 min to isolate serum. Soluble Flt-1 (sFlt-1/VEGF-R1) levels were quantified using a mouse ELISA kit (R\u0026amp;D Systems, #MVR100, Minneapolis, Minnesota) with a sensitivity of 15.2 pg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample Size Determination for Murine Experiments\u003c/strong\u003e. To determine the number of BPH/5 mice required to detect a physiologically meaningful improvement in blood pressure following Ad-hIGF administration, we performed a paired-design sample-size calculation based on repeated pre- and post-injection measurements within the same mouse. Baseline systolic blood pressure in pregnant BPH/5 mice is typically 130\u0026ndash;150 mmHg, with a within-group standard deviation of ~12 mmHg. Assuming a within-animal correlation of r = 0.7 between pre- and post-treatment measurements, the standard deviation of the paired differences is approximately 9.3 mmHg. For a biologically relevant 10 mmHg decrease in systolic blood pressure after Ad-hIGF treatment, the paired standardized effect size (Cohen\u0026rsquo;s d) is ~1.08, representing a very large within-animal effect. Using a two-sided \u0026alpha; = 0.05, power = 80\u0026ndash;90%, the required number of paired animals is 7.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u0026nbsp;\u003c/strong\u003eContinuous outcomes (e.g., longitudinal blood pressure and changes in blood pressure, weight, histologic scores, and morphometric measures) were analyzed separately for BPH/5 and C57BL/6 mice, when applicable, using linear mixed-effects models to compare treatment groups while accounting for correlations among repeated or clustered observations. Models included treatment as a fixed effect and incorporated additional prespecified covariates as appropriate (e.g., time and treatment-by-time interaction for longitudinal outcomes). Random intercepts were included to account for within-individual dependency (e.g., mouse-level random intercept for repeated measures; placenta- or litter-level random intercept for biological replicates). Model-based group estimates are reported as estimated marginal means with 95% confidence intervals.\u003c/p\u003e\n\u003cp\u003eBinary outcomes, including live birth, fetal demise, and resorption, were analyzed using binomial regression models (logit link) with treatment as the primary predictor, fitted separately by strain when applicable. Descriptive probabilities were summarized at the litter level, and model-based mean probabilities were obtained from the fitted binomial models. Between-group comparisons were conducted based on odds ratios (ORs) with corresponding 95% confidence intervals. Multiple testing was addressed using Tukey\u0026rsquo;s significant difference adjustment for pairwise contrasts. All tests were two-sided with statistical significance assessed at \u0026alpha; = 0.05. Modeling analyses were performed in R (version 4.5.1) using \u0026lsquo;lme4\u0026rsquo; and \u0026lsquo;lmerTest\u0026rsquo; for mixed-effects modeling, \u0026lsquo;stats\u0026rsquo; for binomial modeling, and \u0026lsquo;emmeans\u0026rsquo; for marginal means and multiplicity-adjusted contrasts.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eQuantitative analyses for Fig 5-9 were conducted using GraphPad Prism (v9.0 or later; GraphPad Software, San Diego, CA). Data are presented as mean \u0026plusmn; standard deviation (SD) or median \u0026plusmn; IQR where appropriate. For comparisons between two groups, an unpaired two-tailed Student\u0026rsquo;s t-test or Mann-Whitney U was applied where appropriate. In cases involving two independent variables (e.g., treatment and oxygen condition), a two-way ANOVA with Bonferroni correction was conducted. For the in vitro angiogenesis assay, node and mesh counts were quantified from Geltrex-coated 96-well plates using the ImageJ angiogenesis analyzer plugin. For the Ki-67 proliferation assay, flow cytometry data were analyzed using FlowJo software, with Ki-67 positivity quantified as the percentage of PE-positive cells in each treatment group. *P\u0026thinsp;\u0026lt;0.05, **P \u0026lt;\u0026thinsp;0.01; ***P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ****P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ns, not significant. Exact \u003cem\u003ep\u003c/em\u003e-values and details of statistical tests used are reported in figure legends.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIntraplacental gene transfer of IGF-1 at e16 corrects preeclampsia in the BPH/5 mouse. Gene transfer occurred when symptoms of preeclampsia had already begun to manifest in BPH/5 mice. Collectively, the diastolic, systolic, and mean arterial blood pressures all normalized after IGF-1 gene transfer to the levels observed in C57BL/6 controls. BPH/5 mice treated with Ad-hIGF-1 had significantly lower urinary protein than non-pregnant BPH/5 mice and a trend toward lower urinary protein compared with BPH/5-Ad-lacZ-treated mice. The glomerulosclerosis index was also reduced by 65% compared to BPH/5 Ad-LacZ-treated mice and by 55% compared to BPH/5 surgical sham mice. Importantly, intraplacental gene transfer with Ad-hIGF-1 did not compromise fetal outcomes, with no change in the number of live births, rate of demise, or reabsorption.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo validate the microvascular histologic changes observed in BPH/5 placentas, we analyzed a human placenta cohort of pregnancies complicated by early-onset preeclampsia (EOPE), finding a significant alteration in the placental villous morphology and vascularization compared to healthy and a gestational age-matched PPROM control. EOPE placentas demonstrated a reduction in microvascular density, lumens per villous, and maximum villous diameter, and an increase in total syncytial knots. The chorionic villous count did not differ between EOPE vs. PPROM. There was no difference in minimum villous diameter. The changes in placental morphometric microvascular architecture mirrored those found in BPH/5 mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBPH/5 mice had evidence of poor vascularization and vessel architecture. Microvascular density in the BPH/5 mice was lowest in the BPH/5-Ad-lacZ-treated and BPH/5 surgical sham groups. BPH/5 mice treated with Ad-hIGF-1 and BPH/5 surgical sham mice exhibited significantly higher microvascular density, restoring levels of placental vascular integrity that became indistinguishable from those of C57BL/6 controls.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMechanistically, we found IGF-1 activated the IKK\u0026beta;/NF-\u0026kappa;B signaling axis in human microvascular placental endothelial cells, driving angiogenesis and cell proliferation. HPVECs treated with IGF-1 in the presence of pharmacologic inhibitors targeting various blocking steps within the IKK\u0026beta;/NF-\u0026kappa;B, mTOR, PI3K, and AKT pathways found that under normoxic and hypoxic conditions, IGF-1 increased tube formation, which was blocked by IKK\u0026beta;/NF-\u0026kappa;B inhibition and, to a lesser extent, mTOR inhibition. Furthermore, angiogenesis and proliferation were restored in HPVECs under serum-free normoxic and hypoxic conditions under stimulation with IGF-1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo evaluate whether IGF-1 stimulated NF-kB binding of angiogenic gene promoters\u0026rsquo; regions through ChIP-qPCR, we found that in BeWo cells, IGF-1 promotes NF-\u0026kappa;B p65 occupancy at key VEGFA, PlGF, and HIF-1a\u0026nbsp;promoter regions. However, these findings were not seen in HPVECs. These findings suggest that the previously observed angiogenesis, proliferation, and signal-transduction effects indicate that NF-\u0026kappa;B activity within HPVECs may act through other angiogenic gene promoter regions not assessed in these assays, and/or that cell\u0026ndash;cell crosstalk between syncytiotrophoblasts and microvascular endothelial cells contributes to the angiogenic effects observed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;It is surprising that IGF-1 gene transfer did not rescue fetal growth restriction in the BPH/5 mouse. Previously, IGF-1 placental gene transfer has been shown to correct birth weight in the MUAL model of fetal growth restriction\u003csup\u003e29\u003c/sup\u003e as well as in the naturally occurring FGR model in the rabbit.\u003csup\u003e28\u003c/sup\u003e IGF-1 has been shown not only to increase microvascular density but also to increase GLUT transporter expression and localization in both mouse models of FGR and human BeWo cells.\u003csup\u003e29\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;While IGF-1 corrected the preeclampsia phenotype in the BPH/5 mice, IGF-1 may fail to rescue FGR because the BPH/5 placenta may not appropriately respond to IGF-1, resulting in little or no upregulation of nutrient transporters. Even if IGF-1 impacts nutrient transporters in the BPH/5 mice, it may be that early defective placental development impacts their ability to appropriately respond. Additionally, the BPH/5 model notoriously has smaller litter sizes compared to C57BL/6 mice. This surprising lack of IGF-1 to correct FGR requires further investigation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere are additional limitations that warrant consideration. While the BPH/5 model recapitulates many features of preeclampsia, it does not fully capture the complexity of human disease. While IGF-1 activates NF-\u0026kappa;B in trophoblasts and microvascular placental endothelial cells, alternate cellular crosstalk was not explored. Furthermore, while our findings indicate that the IGF-1 signal transduction pathway is involved in preeclampsia, we recognize that multiple phenotypes of human preeclampsia may exist. Given our prior research supporting placental gene transfer of IGF-1 corrected growth in both rabbit and mouse models of FGR, we surmise that IGF-1 is likely involved in early onset phenotypes of preeclampsia that often co-occur with FGR.\u003csup\u003e28,29,43\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAdditionally, our study did not find differences in sFlt-1 levels in the BPH/5 model after IGF-1 transfer. While tremendous clinical focus has centered on the downstream biomarkers like sFlt-1, this may represent outlier readouts rather than faithful indicators of mechanistic activity, especially in early-onset phenotypes. This observation is not new, as previous studies have not found that sFLt-1 was not elevated in the BPH/5 model. The placental microvascular density changes observed within our EOPE cohort were compared to those of healthy patients with differences in gestational age, which may impact vascular density. However, when compared to the PPROM patient, the results were comparable. In addition, adenovirus, while an efficient experimental means of gene transfer, is not an ideal vector for clinical application of placental gene therapy due to the inflammatory response it elicits. \u0026nbsp;Clinical translation of placental gene transfer of IGF-1 will require the development of safer gene transfer platforms, such as adeno-associated virus or nanoparticles.\u003c/p\u003e\n\u003cp\u003eWhile the results of IGF-1 intraplacental gene transfer in correcting preeclampsia in BPH/5 mice are encouraging, the implications for treating human diseases are uncertain. Further exploration of IGF-1 in preeclampsia is warranted before clinical translation can be considered. Preeclampsia continues to remain a leading cause of maternal and perinatal morbidity and mortality, with no current therapeutic options available due to an incomplete understanding of its multifactorial pathogenesis. Our observations highlight IGF-1-mediated NF-\u0026kappa;B activation as an area of investigation and as a potential therapeutic strategy. IGF-1 restores placental angiogenesis, reduces maternal hypertension, and prevents end-organ dysfunction, while preserving maternal and neonatal outcomes in BPH/5 mice. These findings define a mechanistic link between IGF-1 signaling, endothelial function, and placental vascular health, providing a foundation for future investigation to prevent or treat severe preeclampsia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e The data have been made private due to privacy reasons in human subject research and can be made available upon reasonable request to the authors. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e The authors report no conflicts of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors report funding for this research from the Connecticut Children\u0026rsquo;s Connection Grant.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We would like to acknowledge Dr. Jennifer Sones for her collaboration and gift of the BPH/5 line to allow this research to occur\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions (CRediT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization \u0026ndash; KLM, SM, TMC\u003c/p\u003e\n\u003cp\u003eData Curation \u0026ndash; KLM, SM\u003c/p\u003e\n\u003cp\u003eFormal Analysis \u0026ndash; CLK, KLM, SM\u003c/p\u003e\n\u003cp\u003eFunding Acquisition \u0026ndash; SM, TMC\u003c/p\u003e\n\u003cp\u003eInvestigation \u0026ndash; AK, KLM, SM, TMC\u003c/p\u003e\n\u003cp\u003eMethodology \u0026ndash; KLM, TMC\u003c/p\u003e\n\u003cp\u003eProject Administration \u0026ndash; TMC\u003c/p\u003e\n\u003cp\u003eResources \u0026ndash; JS, TMC\u003c/p\u003e\n\u003cp\u003eSoftware \u0026ndash; CLK, SM, KLM\u003c/p\u003e\n\u003cp\u003eSupervision \u0026ndash; JS, TMC\u003c/p\u003e\n\u003cp\u003eValidation \u0026ndash; KLM, SM, TMC\u003c/p\u003e\n\u003cp\u003eVisualization \u0026ndash; KLM, SM, CLK, TMC\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Original Draft \u0026ndash; KLM, TMC\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; Review \u0026amp; Editing \u0026ndash; KLM, SM, CLK, JS, AK, TM\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eChau, K., Welsh, M., Makris, A. \u0026amp; Hennessy, A. Progress in preeclampsia: the contribution of animal models. \u003cem\u003eJ Hum Hypertens\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e, 705-710 (2022).\u003c/li\u003e\n\u003cli\u003eGestational Hypertension and Preeclampsia: ACOG Practice Bulletin, Number 222. \u003cem\u003eObstet Gynecol\u003c/em\u003e \u003cstrong\u003e135\u003c/strong\u003e, e237-e260 (2020).\u003c/li\u003e\n\u003cli\u003eGoldenberg, R.L., Culhane, J.F., Iams, J.D. \u0026amp; Romero, R. Epidemiology and causes of preterm birth. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e371\u003c/strong\u003e, 75-84 (2008).\u003c/li\u003e\n\u003cli\u003eChappell, L.C., Cluver, C.A., Kingdom, J. \u0026amp; Tong, S. Pre-eclampsia. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e398\u003c/strong\u003e, 341-354 (2021).\u003c/li\u003e\n\u003cli\u003eAvorgbedor, F.\u003cem\u003e, et al.\u003c/em\u003e Hypertension and infant outcomes: North Carolina pregnancy risks assessment monitoring system data. \u003cem\u003ePregnancy Hypertens\u003c/em\u003e \u003cstrong\u003e28\u003c/strong\u003e, 189-193 (2022).\u003c/li\u003e\n\u003cli\u003eHenderson, J.T.\u003cem\u003e, et al.\u003c/em\u003e Low-dose aspirin for prevention of morbidity and mortality from preeclampsia: a systematic evidence review for the U.S. Preventive Services Task Force. \u003cem\u003eAnn Intern Med\u003c/em\u003e \u003cstrong\u003e160\u003c/strong\u003e, 695-703 (2014).\u003c/li\u003e\n\u003cli\u003eSrinivas, S.K.\u003cem\u003e, et al.\u003c/em\u003e Rethinking IUGR in preeclampsia: dependent or independent of maternal hypertension? \u003cem\u003eJ Perinatol\u003c/em\u003e \u003cstrong\u003e29\u003c/strong\u003e, 680-684 (2009).\u003c/li\u003e\n\u003cli\u003eRobertson, S.A. Preventing Preeclampsia by Silencing Soluble Flt-1? \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e380\u003c/strong\u003e, 1080-1082 (2019).\u003c/li\u003e\n\u003cli\u003eAubuchon, M., Schulz, L.C. \u0026amp; Schust, D.J. Preeclampsia: animal models for a human cure. \u003cem\u003eProc Natl Acad Sci U S A\u003c/em\u003e \u003cstrong\u003e108\u003c/strong\u003e, 1197-1198 (2011).\u003c/li\u003e\n\u003cli\u003eGatford, K.L., Andraweera, P.H., Roberts, C.T. \u0026amp; Care, A.S. Animal Models of Preeclampsia: Causes, Consequences, and Interventions. \u003cem\u003eHypertension\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 1363-1381 (2020).\u003c/li\u003e\n\u003cli\u003eBakrania, B.A., George, E.M. \u0026amp; Granger, J.P. Animal models of preeclampsia: investigating pathophysiology and therapeutic targets. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e226\u003c/strong\u003e, S973-S987 (2022).\u003c/li\u003e\n\u003cli\u003eTaylor, E.B. \u0026amp; George, E.M. Animal Models of Preeclampsia: Mechanistic Insights and Promising Therapeutics. \u003cem\u003eEndocrinology\u003c/em\u003e \u003cstrong\u003e163\u003c/strong\u003e(2022).\u003c/li\u003e\n\u003cli\u003eRana, S., Burke, S.D. \u0026amp; Karumanchi, S.A. Imbalances in circulating angiogenic factors in the pathophysiology of preeclampsia and related disorders. \u003cem\u003eAm J Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e226\u003c/strong\u003e, S1019-S1034 (2022).\u003c/li\u003e\n\u003cli\u003eHan, L., Luo, Q.Q., Peng, M.G., Zhang, Y. \u0026amp; Zhu, X.H. miR-483 is downregulated in pre-eclampsia via targeting insulin-like growth factor 1 (IGF1) and regulates the PI3K/Akt/mTOR pathway of endothelial progenitor cells. \u003cem\u003eJ Obstet Gynaecol Res\u003c/em\u003e \u003cstrong\u003e47\u003c/strong\u003e, 63-72 (2021).\u003c/li\u003e\n\u003cli\u003eHe, X.\u003cem\u003e, et al.\u003c/em\u003e The relationship between IGF1 and the expression spectrum of miRNA in the placenta of preeclampsia patients. \u003cem\u003eGinekol Pol\u003c/em\u003e \u003cstrong\u003e90\u003c/strong\u003e, 596-603 (2019).\u003c/li\u003e\n\u003cli\u003eIngec, M., Gursoy, H.G., Yildiz, L., Kumtepe, Y. \u0026amp; Kadanali, S. Serum levels of insulin, IGF-1, and IGFBP-1 in pre-eclampsia and eclampsia. \u003cem\u003eInt J Gynaecol Obstet\u003c/em\u003e \u003cstrong\u003e84\u003c/strong\u003e, 214-219 (2004).\u003c/li\u003e\n\u003cli\u003eUmapathy, A.\u003cem\u003e, et al.\u003c/em\u003e Molecular regulators of defective placental and cardiovascular development in fetal growth restriction. \u003cem\u003eClin Sci (Lond)\u003c/em\u003e \u003cstrong\u003e138\u003c/strong\u003e, 761-775 (2024).\u003c/li\u003e\n\u003cli\u003eMaulik, D., Frances Evans, J. \u0026amp; Ragolia, L. Fetal growth restriction: pathogenic mechanisms. \u003cem\u003eClin Obstet Gynecol\u003c/em\u003e \u003cstrong\u003e49\u003c/strong\u003e, 219-227 (2006).\u003c/li\u003e\n\u003cli\u003eTun, W.M., Yap, C.H., Saw, S.N., James, J.L. \u0026amp; Clark, A.R. Differences in placental capillary shear stress in fetal growth restriction may affect endothelial cell function and vascular network formation. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 9876 (2019).\u003c/li\u003e\n\u003cli\u003eWang, Y. \u0026amp; Zhao, S. in \u003cem\u003eVascular Biology of the Placenta\u003c/em\u003e (San Rafael (CA), 2010).\u003c/li\u003e\n\u003cli\u003eArroyo, J., Torry, R.J. \u0026amp; Torry, D.S. Deferential regulation of placenta growth factor (PlGF)-mediated signal transduction in human primary term trophoblast and endothelial cells. \u003cem\u003ePlacenta\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, 379-386 (2004).\u003c/li\u003e\n\u003cli\u003eLuo, Z.C.\u003cem\u003e, et al.\u003c/em\u003e Maternal and fetal IGF-I and IGF-II levels, fetal growth, and gestational diabetes. \u003cem\u003eJ Clin Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 1720-1728 (2012).\u003c/li\u003e\n\u003cli\u003eHiden, U., Glitzner, E., Hartmann, M. \u0026amp; Desoye, G. Insulin and the IGF system in the human placenta of normal and diabetic pregnancies. \u003cem\u003eJ Anat\u003c/em\u003e \u003cstrong\u003e215\u003c/strong\u003e, 60-68 (2009).\u003c/li\u003e\n\u003cli\u003eNawathe, A.R.\u003cem\u003e, et al.\u003c/em\u003e Insulin-like growth factor axis in pregnancies affected by fetal growth disorders. \u003cem\u003eClin Epigenetics\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 11 (2016).\u003c/li\u003e\n\u003cli\u003eMajumder, S., Moriarty, K.L., Lee, Y. \u0026amp; Crombleholme, T.M. Placental Gene Therapy for Fetal Growth Restriction and Preeclampsia: Preclinical Studies and Prospects for Clinical Application. \u003cem\u003eJ Clin Med\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e(2024).\u003c/li\u003e\n\u003cli\u003eSu, J., Huang, X., Meng, S. \u0026amp; Wang, S. Investigating Serum and Placental Levels of IGF-1 and IGF-1R in Preeclampsia Patients and Their Clinical Implications. \u003cem\u003eInt J Womens Health\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, 729-738 (2025).\u003c/li\u003e\n\u003cli\u003eLiu, J.P., Baker, J., Perkins, A.S., Robertson, E.J. \u0026amp; Efstratiadis, A. Mice carrying null mutations of the genes encoding insulin-like growth factor I (Igf-1) and type 1 IGF receptor (Igf1r). \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e75\u003c/strong\u003e, 59-72 (1993).\u003c/li\u003e\n\u003cli\u003eKeswani, S.G.\u003cem\u003e, et al.\u003c/em\u003e Intraplacental gene therapy with Ad-IGF-1 corrects naturally occurring rabbit model of intrauterine growth restriction. \u003cem\u003eHum Gene Ther\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e, 172-182 (2015).\u003c/li\u003e\n\u003cli\u003eJones, H.N., Crombleholme, T. \u0026amp; Habli, M. Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, e74632 (2013).\u003c/li\u003e\n\u003cli\u003eZhang, X.\u003cem\u003e, et al.\u003c/em\u003e IGF-1 inhibits inflammation and accelerates angiogenesis via Ras/PI3K/IKK/NF-kappaB signaling pathways to promote wound healing. \u003cem\u003eEur J Pharm Sci\u003c/em\u003e, 106847 (2024).\u003c/li\u003e\n\u003cli\u003eSalminen, A. \u0026amp; Kaarniranta, K. Insulin/IGF-1 paradox of aging: regulation via AKT/IKK/NF-kappaB signaling. \u003cem\u003eCell Signal\u003c/em\u003e \u003cstrong\u003e22\u003c/strong\u003e, 573-577 (2010).\u003c/li\u003e\n\u003cli\u003eLiu, P.\u003cem\u003e, et al.\u003c/em\u003e Role and mechanisms of the NF-kB signaling pathway in various developmental processes. \u003cem\u003eBiomed Pharmacother\u003c/em\u003e \u003cstrong\u003e153\u003c/strong\u003e, 113513 (2022).\u003c/li\u003e\n\u003cli\u003eAdli, M., Merkhofer, E., Cogswell, P. \u0026amp; Baldwin, A.S. IKKalpha and IKKbeta each function to regulate NF-kappaB activation in the TNF-induced/canonical pathway. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e9428 (2010).\u003c/li\u003e\n\u003cli\u003eRao, S., Liu, M., Iosef, C., Knutsen, C. \u0026amp; Alvira, C.M. Endothelial-specific loss of IKKbeta disrupts pulmonary endothelial angiogenesis and impairs postnatal lung growth. \u003cem\u003eAm J Physiol Lung Cell Mol Physiol\u003c/em\u003e \u003cstrong\u003e325\u003c/strong\u003e, L299-L313 (2023).\u003c/li\u003e\n\u003cli\u003eIosef, C.\u003cem\u003e, et al.\u003c/em\u003e Inhibiting NF-kappaB in the developing lung disrupts angiogenesis and alveolarization. \u003cem\u003eAm J Physiol Lung Cell Mol Physiol\u003c/em\u003e \u003cstrong\u003e302\u003c/strong\u003e, L1023-1036 (2012).\u003c/li\u003e\n\u003cli\u003eAshida, N.\u003cem\u003e, et al.\u003c/em\u003e IKKbeta regulates essential functions of the vascular endothelium through kinase-dependent and -independent pathways. \u003cem\u003eNat Commun\u003c/em\u003e \u003cstrong\u003e2\u003c/strong\u003e, 318 (2011).\u003c/li\u003e\n\u003cli\u003eDodson, R.B.\u003cem\u003e, et al.\u003c/em\u003e Intrauterine growth restriction decreases NF-kappaB signaling in fetal pulmonary artery endothelial cells of fetal sheep. \u003cem\u003eAm J Physiol Lung Cell Mol Physiol\u003c/em\u003e \u003cstrong\u003e315\u003c/strong\u003e, L348-L359 (2018).\u003c/li\u003e\n\u003cli\u003eDavisson, R.L.\u003cem\u003e, et al.\u003c/em\u003e Discovery of a spontaneous genetic mouse model of preeclampsia. \u003cem\u003eHypertension\u003c/em\u003e \u003cstrong\u003e39\u003c/strong\u003e, 337-342 (2002).\u003c/li\u003e\n\u003cli\u003eSones, J.L., Yarborough, C.C., O\u0026apos;Besso, V., Lemenze, A. \u0026amp; Douglas, N.C. Genotypic analysis of the female BPH/5 mouse, a model of superimposed preeclampsia. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, e0253453 (2021).\u003c/li\u003e\n\u003cli\u003eBurton, G.J.\u003cem\u003e, et al.\u003c/em\u003e Optimising sample collection for placental research. \u003cem\u003ePlacenta\u003c/em\u003e \u003cstrong\u003e35\u003c/strong\u003e, 9-22 (2014).\u003c/li\u003e\n\u003cli\u003eSchindelin, J.\u003cem\u003e, et al.\u003c/em\u003e Fiji: an open-source platform for biological-image analysis. \u003cem\u003eNat Methods\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, 676-682 (2012).\u003c/li\u003e\n\u003cli\u003eKurtz, T.W.\u003cem\u003e, et al.\u003c/em\u003e Recommendations for blood pressure measurement in humans and experimental animals: part 2: blood pressure measurement in experimental animals: a statement for professionals from the Subcommittee of Professional and Public Education of the American Heart Association Council on High Blood Pressure Research. \u003cem\u003eArterioscler Thromb Vasc Biol\u003c/em\u003e \u003cstrong\u003e25\u003c/strong\u003e, e22-33 (2005).\u003c/li\u003e\n\u003cli\u003eHabli, M., Jones, H., Aronow, B., Omar, K. \u0026amp; Crombleholme, T.M. Recapitulation of characteristics of human placental vascular insufficiency in a novel mouse model. \u003cem\u003ePlacenta\u003c/em\u003e \u003cstrong\u003e34\u003c/strong\u003e, 1150-1158 (2013).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8563004/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8563004/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eIntroduction. \u003c/strong\u003ePreeclampsia causes severe complications for the mother, fetus, and newborn, yet the underlying mechanisms remain poorly understood. The BPH/5 mouse is the only spontaneous mouse model that recapitulates key features of human preeclampsia. Although impaired angiogenesis and endothelial dysfunction are hallmarks of this disease, the molecular pathways capable of restoring placental vascular integrity remain undefined. Existing animal models of preeclampsia have not directly targeted the placenta. We hypothesized that hIGF-1 rescues placental endothelial function by driving angiogenic gene expression through the IKK-β/NF-κB signaling axis, thereby correcting the maternal pathophysiologic features of preeclampsia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods. \u003c/strong\u003ePlacental morphometric analysis for CD31 immunostaining was performed on human placental samples from early onset preeclampsia (EOPE), gestational age-matched preterm premature rupture of membranes (PPROM), and term healthy controls. Next, primary human placental microvascular endothelial cells were cultured to evaluate IGF-1–mediated responses using in vitro angiogenesis assays under normoxic and hypoxic conditions, in the presence or absence of established NF-κB inhibitors. Cell proliferation was assessed using Ki-67 immunostaining and flow cytometry, and PCR-chromatin immunoprecipitation was used to quantify NF-κB binding to promoter regions of angiogenic genes in human placenta vascular endothelial cells as well as BeWo cells. In parallel, BPH/5 and C57BL/6 mice were time-mated and habituated to blood pressure cuff monitoring. Intraplacental gene delivery of 1x10\u003csup\u003e8\u003c/sup\u003e PFU of Ad-hIGF-1 (referred to as Ad-IGF-1) or Ad-LacZ was performed on embryonic day 16 with cerclage, followed by harvest on e21. Maternal endpoints of blood pressure and proteinuria were assessed at non-pregnant, first-trimester, pre-injection, and post-injection time points. Kidney histology, sFlt-1 levels, and placental endothelial microvascular density assessment were evaluated. Fetal endpoints included litter outcomes.\u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults. \u003c/strong\u003eMorphometric placental analysis of EOPE vs. term healthy and PPROM controls showed that microvascular density is markedly reduced while villous architecture remains preserved. BPH/5 placentas similarly exhibit reduced microvascular density in comparison to C57BL/6 placentas. Restoration of microvasculature was appreciated after IGF-1 gene transfer. Angiogenesis and proliferation assays in HPVECs demonstrated that IGF-1 robustly enhances both angiogenic activity and cell proliferation under normoxic and hypoxic conditions, primarily through IKKβ/NF-κB–dependent transcriptional activation of key angiogenic genes. Interestingly, IGF-1 was found to enhance NF-κB signal transduction of angiogenic gene promoters in BeWo cells, but not HPVECs.\u003c/p\u003e\n\u003cp\u003eIn the BPH/5 mouse, intraplacental gene transfer of IGF-1 reduced the post-injection systolic, diastolic, and mean arterial pressures comparable to C57BL/6 controls, with the SBP consistently reduced at all delta comparisons across timepoints. Urinary protein levels in BPH/5 were also comparable to controls after gene transfer with Ad-IGF-1. Litter size, demise rate, reabsorptions, and pup weight were unaffected by Ad-IGF-1 gene transfer. Ad-IGF-1 treatment reduced glomerulosclerosis (47.2% vs. surgical sham; 58% vs. Ad-LacZ controls) while liver histology and s-Flt-1 were unchanged.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion. \u003c/strong\u003eIGF-1 gene transfer reverses the preeclampsia-like phenotype in BPH/5 mice by restoring placental microvascular density without affecting fetal outcomes in the first animal model of preeclampsia treatment that directly targets the placenta. Furthermore, IGF-1’s pro-angiogenic effects are suggested to occur via IKKβ/NF-κB–dependent activation.\u003c/p\u003e","manuscriptTitle":"Correction of Preeclampsia by Intraplacental Gene Transfer of IGF-1 in the BPH/5 Mouse via NF-KB Mediated Induction of Angiogenic Gene Expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-16 11:24:25","doi":"10.21203/rs.3.rs-8563004/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"18708c65-eac6-42a0-8277-a90905d1a683","owner":[],"postedDate":"January 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61022767,"name":"Health sciences/Diseases/Reproductive disorders"},{"id":61022768,"name":"Biological sciences/Biotechnology/Gene therapy"},{"id":61022769,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":61022770,"name":"Biological sciences/Physiology/Cardiovascular biology/Cardiovascular diseases/Hypertension/Pre-eclampsia"}],"tags":[],"updatedAt":"2026-01-16T11:24:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-16 11:24:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8563004","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8563004","identity":"rs-8563004","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}