Losartan shows limited benefit in preclinical models of Geleophysic dysplasia

Losartan shows limited benefit in preclinical models of Geleophysic dysplasia | 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 Losartan shows limited benefit in preclinical models of Geleophysic dysplasia Alejo Antonio Morales, Vladimir Camarena, LéShon Peart, Sarah Smithson, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8301608/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Geleophysic dysplasia (GD) is a rare genetic disorder characterized by short stature, joint contractures, and cardiopulmonary complications, with early mortality, and linked to mutations in ADAMTSL2 (GD1), FBN1 (GD2), or LTBP3 (GD3) genes. These mutations are hypothesized to disrupt extracellular matrix (ECM) organization and enhance transforming growth factor beta (TGF-β) signaling. Losartan, an angiotensin II receptor blocker, has been proposed to mitigate TGF-β-mediated pathologies. In this study we tested the efficacy of losartan as a therapeutic drug for GD. We evaluated losartan's therapeutic potential using ADAMTSL2 p.A165T mutant mice and patient-derived fibroblasts. Survival, growth, TGF-β signaling, and ECM protein expression were assessed. Losartan did not improve survival or growth in mutant mice. Patient fibroblasts exhibited reduced basal TGF-β1 secretion and SMAD phosphorylation without transcriptomic evidence of pathway activation. Losartan treatment failed to modulate TGF-β signaling or ECM protein incorporation. These results suggest limited benefits of losartan in GD and challenge the notion of TGF-β dysregulation in GD pathogenesis, indicating a need for alternative targeted therapies. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Genetics Health sciences/Medical research Biological sciences/Molecular biology Geleophysic dysplasia ADAMTSL2 FBN1 TGF-β1 losartan extracellular matrix Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Geleophysic dysplasia (GD) is a progressive genetic disorder presenting with short stature, short fingers and toes, joint contractures, distinctive facial features, and cardiopulmonary complications that contribute to poor prognosis. 1–7 To date, three genes have been linked to GD: Geleophysic dysplasia type 1 (GD1, GPHYSD1, OMIM 231050) is an autosomal recessive form caused by mutations in ADAMTSL2 (OMIM 612277). 8 GD type 2 (GD2, GPHYSD2, OMIM 614185) is autosomal dominantly inherited resulting from mutations in exons 41 or 42 of FBN1 (OMIM 134797). 9 GD type 3 (GD3, GPHYSD3, OMIM 614185) is also autosomal dominant and caused by variants in LTBP3 (OMIM 602090). 10 ADAMTSL2 encodes a secreted matricellular glycoprotein within the ADAMTS ( A D isintegrin A nd M etalloprotease with T hrombospondin motifs) superfamily, but it lacks the metalloprotease domain typical of this group. 11–13 Although the function of ADAMTSL2 is not fully understood, emerging evidence points to its role in organizing fibrillin microfibrils 14 – 17 and modulating the availability of Transforming Growth Factor Beta (TGF-β) via interaction with extracellular matrix (ECM) proteins such as FNB1 and LTBP1. 8,9,18–21 Fibrillin-1, encoded by the FBN1 gene, is a large, calcium-binding ECM glycoprotein and a key structural component of microfibrils, with a regulatory role in the bioavailability of molecules like TGF-β. 20–24 FBN1 mutations can cause Marfan syndrome, 25,26 but variants, in exons 41–42, which encode the TGF-β-binding protein-like domain 5 (TB5), can result in GD2. 8,9,26 These mutations are thought to contribute to GD2 through disruption of TGF-β signaling. Latent Transforming Growth Factor Beta Binding Protein 3 (LTBP3) is an extracellular matrix protein that regulates the availability of TGF-β. 27,28 Variants in LTBP3 have been linked to genetic disorders with skeletal dysplasia, including GD3. 10,29,30 Four major mechanisms have been proposed in GD pathogenesis: First, mutations in ADAMTSL2 , FBN1 and LTBP3 genes have been shown to impair protein secretion disrupting the ECM organization. 8,28,31–33 Second, an increased TGF-β activity has been observed in cells from individuals with GD. 8–10,15,16,18,34,35 Third, our recent findings implicate matrix metalloproteinases (MMPs), which are a family of enzymes responsible for ECM remodeling, as potential contributors to GD pathology. 36 Four, the mutations cause a reduction in the Wnt signaling pathway. 37 A clear genotype-phenotype correlation in patients with GD has not yet been established. However, our recent work demonstrates that the clinical severity of GD1 correlates with the abundance of ADAMTSL2 in the extracellular matrix. 31 Using both cellular and murine models that replicate the genetic profile of a GD patient compound heterozygous for two ADAMTSL2 variants, p.R61H and p.A165T, we observed the impaired secretion of ADAMTSL2 in patient-derived dermal fibroblasts and in HEK-293T overexpression systems. 31 Notably, the p.A165T variant resulted in a more pronounced secretion defect. 31 Mice homozygous or hemizygous for the p.A165T variant exhibited growth impairment, respiratory and cardiac dysfunction, and early mortality. 31 Losartan, an angiotensin II type 1 receptor blocker, has been proposed to ameliorate Marfan syndrome pathology through inhibition of TGF-β signaling. 38 Losartan could hypothetically offer therapeutic benefit in GD by modulating TGF-β pathway. In this study, we evaluated the effects of losartan in both in-vivo and in-vitro models of GD1, using optimized dosing strategies in a mouse model carrying the p.A165T ADAMTSL2 variant, as well as in patient-derived fibroblasts. Our goal was to determine whether losartan could improve survival or growth in the animal model or restore the ECM organization in patient cells. Results Losartan treatment does not improve the survival of Adamtsl2 mutant mice To test the effects of losartan in-vivo , we utilized our previously generated GD animal models. Mice that were hemizygous for the Adamtsl2 p.A165T missense variant were selected for treatment due to their phenotypic severity, especially their reduced survival and stunted growth. 31 Initially, we followed the treatment scheme and dosage previously used in mouse models of Marfan syndrome, consisting of prenatal and postnatal losartan treatments of 0.6 g/liter in the drinking water. 39 We found an increased lethality in the offspring of mice regardless of genotype with less than 40% survival in the first month ( Figure S1 A ). This treatment scheme was not continued because of the high toxicity in WT mice. The toxicity of losartan for the offspring during gestation is well documented; it can cross the placenta barrier, and it is secreted with the milk. For these reasons, losartan is a category D pregnancy risk medication and contraindicated in pregnant women. 40 – 44 Such toxicity was not found in previous studies with the Marfan syndrome mouse models; a possible explanation could be variations in the water intake among mice strains. 45,46 An adult mouse depending on the strain can drink from 2.5 to 50 mL of water per day. 45,46 Adult C57BL6 female mice with 25 g of weight, the same strain of our GD mice, drink around 6.4 mL water per day. 45 Most of the toxicity studies in animals have been done on rats. The maximum dose in rats was calculated to be 10 to 20 mg/kg/day, and in humans 1 to 3 mg/kg/day. 40,47 In mice, previous studies have used losartan within 10 to 42.5 mg/kg/day. 40,48,49 Based on this information, we calculated that a dose 0.6 g/L in the drinking water provides 153 mg/kg/day of losartan in our animals, which is well above the estimated maximum dose. We proceeded to do a dose curve with our WT animals to determine the dose of losartan (from E18 to postnatal day 20) that could be tolerated without a significant effect in survival ( Figure S1 B ) and found that 0.15 g/L in their drinking water provides a 38.25 mg/kg/day of losartan and has a minimal toxicity ( Figure S1 B ). Therefore, we used this optimized dose to explore the potential therapeutic effect of losartan in the GD animal models. Our previous data show that p.A165T hemizygous mice (A165T/-) present with a significant reduction in survival (~ 40%) after 3 months. 31 Most of the lethality was observed in the first few days after birth. When we treated mice with the losartan dose of 38.5 mg/kg/day from E18, we did not find a significant improvement in survival in the p.A165T hemizygous offspring (Fig. 1 A). Weight was monitored on days 5.5, 15.5, 22.5, and 36.5. Hemizygous p.A165T animals showed significantly reduced weight, starting from 15.5 days of age in males and females, when compared to WT, regardless of the treatment status ( p < 0.001, p < 0.001, and p < 0.001 in treated p.A165T hemizygous males at 15.5, 22.5, and 36.5 days, p = 0.07, p < 0.001, and p = 0.002 in treated females p.A165T hemizygous at 15.5, 22.5, and 36.5 days) (Fig. 1 B). These results indicate that using losartan in this treatment modality does not have a beneficial effect on the survival or growth of GD1 mouse models. At the end of all experiments, animals were euthanized using the carbon dioxide protocol and then cervical dislocation, as approved by our Institutional Animal Care and Use Committee (IACUC) and according to the National Institutes of Health guidelines. Fibroblasts from patients with GD1 and GD2 Previous studies, including ours, have shown that mutations in the ADAMTSL2 and FBN1 genes impair the secretion of these proteins. 8,9,12,31 GD006 patient fibroblasts (GD1-2) were kindly provided by the Bristol Genetics Laboratory at North Bristol NHS Trust, Severn Pathology, Southmead Hospital, UK. To further investigate the cellular and molecular consequences of these variants, we analyzed primary human dermal fibroblasts obtained from patients with ADAMTSL2 or FBN1 mutations. Fibroblasts from healthy individuals were used as controls. Individuals with LTBP3-related GD (GD3) were excluded from this study due to the rarity of this subtype, which accounts for less than 1% of the GD cases. 1 All fibroblasts were isolated using the explant technique, as previously described. 31,36 The gene variants identified in the fibroblast samples are summarized in Table 1 and were confirmed by sequencing of the ADAMTSL2 and FBN1 genes. GD1 patients carried compound heterozygous mutations in the ADAMTSL2 gene, while GD2 patients had heterozygous mutations in the FBN1 gene. Table 1 Details of studied samples. Sample ID CT-1 CT-2 GD1-1 GD1-2 GD2-1 GD2-2 GD2-3 Diagnosis CT CT GD1 GD1 GD2 GD2 GD2 Patient ID GD016E GD017E GD001 GD006 GD004 GD009 GD012 Mutations ADAMTSL2 c.182G > A ( p.R61H) c.493G > A ( p.A165T) ADAMTSL2 c.542T > C ( p.V181A) c.707C > T ( p.P236L) FBN1 c.5284G > A ( p.G1762S) FBN1 c.5183C > T ( p.A1728V) FBN1 c.5243G > C ( p.C1748S) compound heterozygous compound heterozygous heterozygous heterozygous heterozygous Sex male male male female female male male Age - years (months) 0 (1) 0 (6) 1 (11) 12 (0) 28 (9) 8 (8) 11 (0) CT: Healthy control, GD1: Geleophysic Dysplasia type 1, GD2: Geleophysic Dysplasia type 2. GD fibroblasts do not show activation of TGF-β pathway To assess TGF-β production, we used an ELISA assay under three different culture conditions using DMEM medium as the base: 10% FBS, 1% FBS, and serum-free (0% FBS). Although all conditions yielded similar trends, we chose the serum-free condition for subsequent analyses to eliminate potential cross-reactivity from bovine TGF-β and to evaluate TGF-β1 secretion without external stimulation. On average, the active form of TGF-β1 comprised of only about 1% of total TGF-β1 secreted from the cells (Fig. 2 A). The control cells secreted approximately 200 pg/mL over three days. In comparison, patient cells secreted an average of 114 pg/mL, indicating that fibroblasts from patients secreted significantly less amount of total TGF-β1 than controls. We next examined downstream TGF-β signaling by assessing SMAD2 and SMAD3 phosphorylation. Consistent with the low levels of active TGF-β, phosphorylated SMAD2 (S465/467) and SMAD3 (S423/425) were not detected (Fig. 2 B, 2 C and Figure S2) . Total SMAD2 was at similar levels between control and patient cells, while patient fibroblasts showed slightly reduced levels of total SMAD3 and SMAD4 (Fig. 2 B, 2 C). Phosphorylated SMAD1/5/8 (S463/465) was also undetectable in all samples, and total SMAD1 levels did not differ between the two groups (Fig. 2 B, 2 C). To get a deeper insight into TGF-β pathway on patient cells, we analyzed the fibroblast RNA-Seq data to profile the cell transcriptome and to identify active genes and pathways. We identified all genes associated with the TGF-β signaling pathway based on the Gene Set Enrichment Analysis database (GSEA, see methods). The transcript expression for all these selected genes was analyzed from the RNA-Seq data from control and patient-derived fibroblasts. The gene clustering did not reveal any significant dysregulation of the TGF-β pathway members between groups. Among the 56 genes analyzed, none showed a ≥ 2-fold change in expression between patient cells and controls ( Figure S4 ). Detailed transcript expressions for all genes are shown in Table S1 . In a similar way, we next examined genes previously reported to be upregulated following TGF-β1 stimulation (GSEA) and the RNA-Seq data for our cells was analyzed. The gene clustering did not indicate a consistent upregulation of these genes in patient samples ( Figure S5 ). Of the 116 genes evaluated, only one gene, ZIC4 , was significantly upregulated in patient cells ( Table S2 ). Conversely, three genes, CFAP298 , SLC8A1 , and JADE1 , were significantly downregulated (≥ 2-fold), arguing against a generalized activation of TGF-β signaling ( Table S2 ). Finally, we also analyzed genes reported to be downregulated in response to TGF-β stimulation (GSEA). No clear pattern of downregulation emerged from gene clustering and shown in Figure S6 . Among the 176 genes assessed, 10 ( LAMA5 , GATA6 , TGM2 , PLAT , SSX2IP , SYNE2 , C4B , EZR , MET , and ALCAM ) were significantly downregulated in patient cells, while one gene, MTUS1 , was significantly upregulated ( Table S3 ). Collectively, these analyses do not support the hypothesis of TGF-β pathway activation in the patient-derived fibroblasts. To evaluate whether patient fibroblasts retained the ability to respond to TGF-β, we treated both control and patient cells with exogenous TGF-β1 for 1 and 24 hours, then assessed SMAD2 phosphorylation at the S465/467 site, a key event in TGF-β signaling. As shown in Fig. 2 D and Figure S3 , all patient cells responded robustly to TGF-β after 1 hour of stimulation, showing comparable levels of SMAD2 phosphorylation to controls. Similar results were observed after 24 hours (data not shown), indicating that TGF-β signaling pathway remains intact in patients` fibroblasts despite their reduced basal secretion of TGF-β1 or phosphorylation status of SMAD2 or SMAD3. Stimulation of TGF-β signaling increases ADAMTSL2, FBN1 and fibronectin in fibroblasts To investigate the role of the TGF-β signaling pathway in the regulation of GD-associated proteins, we treated control and patient-derived fibroblasts with TGF-β1 or BMP2 for seven days and then assessed the levels of ADAMTSL2, FBN1, and fibronectin. To check the pathway activation, we measured SMAD2 phosphorylation in response to TGF-β1 treatment (Fig. 3 and Figure S7 ). TGF-β1 not only induced phosphorylation of SMAD2 but also the phosphorylation of Smad1/5/8, indicating the activation of both the canonical and non-canonical pathways, as previously reported. 50,51 In contrast, BMP2 selectively induced phosphorylation of Smad1/5/8, consistent with its known signaling pathway (Fig. 3 ). TGF-β1 treatment led to a marked upregulation of ADAMTSL2 expression in all patient fibroblasts and in one control line, with increases observed both intracellularly and extracellularly (Fig. 3 ). BMP2 also increased ADAMTSL2 expression, albeit to a lesser extent and only in a subset of the cells tested. Similarly, TGF-β1 increased FBN1 expression in both patient and control fibroblasts, while BMP2 elicited a response only in control cells (Fig. 3 ). Notably, the fold increase in FBN1 expression was greater in the patient fibroblasts, likely due to their lower baseline levels, as we previously described. 31,36 Fibronectin levels were elevated following TGF-β1 treatment in both patient and control fibroblasts, whereas BMP2 had no significant effect (Fig. 3 ). All these results highlight the key role of TGF-β pathway in the ECM organization and suggest that uncontrolled inhibition of the pathway might lead to additional ECM dysfunction. Losartan does not affect TGF-β signaling or ECM protein incorporation in patient fibroblasts To investigate the effect of losartan on TGF-β1 signaling, patient-derived fibroblasts were treated with varying concentrations of the drug. As shown in Fig. 4 A and Figure S8 , losartan did not significantly alter total TGF-β1 levels in the conditioned media under 10% FBS conditions when compared to untreated controls. A slight increase in active TGF-β1 was observed at certain concentrations; however, these values remained below the detection limit of the ELISA and were therefore considered unreliable. At low concentrations, losartan did not affect the total TGF-β1 secretion in patient fibroblasts. Interestingly, treatment with losartan at 300 µM, resulted in a slight but significant increase in total TGF-β1 secretion in patient cells. Despite this increase, no differences in SMAD2 phosphorylation were observed upon losartan treatment, as demonstrated by Western blot analysis (Fig. 4 B), suggesting that downstream TGF-β signaling remained unaltered. Consistent with our previous findings, the ADAMTSL2 protein was predominantly detected in the intracellular (IC) compartment (bands-1 and band-2, Fig. 4 B) with limited detection in the ECM fraction (bands-3 and band-4), relative to IC levels. 31,36 GAPDH and fibronectin served as controls for IC and ECM fractions, respectively. ADAMTSL2 mutations did not affect its intracellular expression, and no significant differences were observed between control and patient fibroblasts. However, secretion of ADAMTSL2 into the conditioned media and its incorporation into the ECM were significantly reduced in patient fibroblasts. Treatment with losartan, even at increasing concentrations, did not alter ADAMTSL2 intracellular expression, secretion, or ECM incorporation (Figs. 4 B, 4 C). In contrast to ADAMTSL2, FBN1 was primarily localized to the ECM, with minimal intracellular localization. 31,36 Notably, the intracellular form of FBN1 exhibited a slightly higher molecular weight compared to its secreted form (Fig. 4 B). As previously reported, ADAMTSL2 mutations led to reduced intracellular FBN1 levels in patient fibroblasts relative to controls (Figs. 4 B, 4 C). Although secretion of FBN1 was modestly affected, differences were not statistically significant. The most pronounced alteration was a substantial reduction in ECM-incorporated FBN1 in patient cells. Losartan treatment did not modify FBN1 intracellular levels, secretion into the conditioned media, or incorporation into the ECM. We further examined the expression and distribution of two additional ECM components, fibronectin and collagen type-I alpha 1 (Col1A1). Fibronectin was primarily detected in the ECM with limited intracellular presence, whereas Col1A1 was largely retained intracellularly with poor ECM incorporation. 31,36 In patient fibroblasts, fibronectin expression and secretion into the conditioned media were comparable to controls; however, ECM incorporation was markedly reduced. Col1A1 showed mildly increased intracellular expression in patient fibroblasts, but secretion levels were unchanged. Like fibronectin, Col1A1 incorporation into the ECM was significantly impaired in the patient cells. Importantly, losartan treatment had no observable effect on the intracellular expression, secretion, or ECM incorporation of either protein (Fig. 4 B, 4 C). Discussion Previous studies have suggested that dysregulation of TGF-β signaling is the underlying mechanism in GD. Fibroblasts derived from patients with mutations in ADAMTSL2 and FBN1 genes have consistently shown altered TGF-β signaling. 1,8,9,20,21,23,24 Although GD-associated mutations for the LTBP3 gene similarly disrupt the microfibrillar network, it does not appear to increase TGF-β signaling in the dermal fibroblast model described for GD3. 1 , 10 Nevertheless, in a different cellular model for adipogenesis, the loss of LTBP3 resulted in a reduced adipogenesis via an increase in TGF beta signaling. 52 In the present study, our ADAMTSL2 p.A165T mouse model displayed severe growth impairment and high perinatal mortality, yet losartan treatment failed to improve either outcome. Unexpectedly, we observed no evidence of TGF-β pathway activation in dermal fibroblasts derived from GD patients. Compared to control fibroblasts, patient cells secreted lower levels of TGF-β1 and lacked detectable SMAD2/3 phosphorylation. Treatment with active TGF-β induced phosphorylation of SMAD2 without significant differences between control and patient cells, suggesting that the signaling machinery remain intact. TGF-β1 and ADAMTSL2 are proposed to be involved in a bidirectional regulatory loop. 15,53 ADAMTSL2 inhibits TGF-β signaling by binding to LTBP1 within the microfibrillar network, thereby reducing the bioavailability of active TGF-β. 15,53 Conversely, TGF-β1 signaling can induce the expression of ADAMTSL2, suggesting a feedback mechanism wherein increased TGF-β activity promotes ADAMTSL2 expression to attenuate further signaling. 18,54 In this study, treatment with TGF-β1 and BMP2 led to increased expression of both ADAMTSL2 and FBN1 in all primary fibroblast. This suggests that upregulation of these ECM components by TGF-β1 is a generalizable mechanism in dermal fibroblasts, independent of the genotype. Our findings suggest that an increase in TGF-β signaling is not the primary driver of disease pathology. In fact, there is evidence from other studies suggesting that an increased TGF-β signaling is not consistently observed across all models of GD. 15,31,37,55 For example, in ADAMTSL2 -deficient myoblasts no increase in TGF-β activity was detected. 37 In Adamtsl2 knockout mice, TGF-β signaling was not elevated at birth and pan-TGF-β neutralizing antibody failed to rescue the phenotype. 15 A similar mechanism involving dysregulated TGF-β signaling has been proposed for Marfan syndrome, which is caused FBN1 mutations. 56–58 Marfan syndrome patients present a phenotype that is notably opposite to that of GD, characterized by tall stature and long extremities. This striking contrast raises the question of how mutations in the same gene and potentially overlapping molecular pathways can lead to such divergent clinical manifestations. Numerous studies have evaluated losartan’s efficacy in Marfan syndrome animal models and patient cohorts, 59–65 yet no investigation has examined its effects in GD patients or animal models. Notably, losartan was reported in 2019 to improve lysosomal inclusions and microfibril deposition in GD2 primary fibroblast in vitro. Motivated by this finding, and by recurrent inquiries from patients and clinicians about losartan as a potential therapy for GD, we investigated its efficacy in our GD models. In our study, losartan conferred no therapeutic benefit in GD1 mice and failed to improve ECM organization in patient primary fibroblasts, suggesting that GD and Marfan syndrome do not share a common pathogenic mechanism. Our findings challenge the assumption that TGF-β dysregulation is a universal feature of GD and suggest that TGF-β-targeted therapies are not effective in GD. Further work is needed to elucidate the precise molecular pathways involved and to identify more targeted therapeutic strategies. Material and Methods Study approval All human and animal studies were reviewed and approved at the University of Miami and by the Institutional Review Board (IRB) and by the Institutional Animal Care and Use Committee (AICUC), respectively, and according to the National Institutes of Health guidelines (NIH). All patients were evaluated at the University of Miami or by collaborators. Written informed consents for the testing and sharing of the research data were obtained from all patients or their parents, including the use of the isolated primary fibroblasts. All animal studies and experiments were performed following all relevant guidelines and regulations; and all reports were in accordance with ARRIVE guidelines. Losartan Potassium Losartan was obtained from Tokyo Chemical Industry, Tokyo Japan. Diluted in the drinking water at 0.6, 0.3, 0.15, and 0.075 g/L to provide 153, 76.5, 38.25, and 19.125 mg/kg/day respectively. Transgenic mice for the generation of Adamtsl2 p.A165T missense variant, we developed a knock-in mouse model carrying the variant by conventional embryonic stem cell-mediated knock-in technology using the service of Taconic-Cyagen Model generation Alliance (Germantown, NY), as previously described. 31 In summary, a targeted C57BL/6J ES cell clone was injected into C57BL/6 albino embryos, Germline transmission was further confirmed after further breading with C57BL6 females. Adamtsl2 p.A165T heterozygous mice were crossed between them to obtain homozygous animals or with heterozygous mice carrying a knock-out allele of Adamtsl2 14 , to obtain hemizygous mice. Animals were genotyped as previously described and housed under a 12-hour light-dark cycle at 20–23°C in specific pathogen-free facilities and supplied with food and water ad libitum. All studies and experiments were performed in accordance with relevant guidelines and regulations. In addition, all reports were in accordance with ARRIVE guidelines. All litters, excluding the first one, from different types of matings, were included in these studies until at least 10 animals per group was reached. Dams with high litter mortality caused by others factor like complications during birth were removed from the study. Adamtsl2 p.A165T hemizygous mice were compared with Adamtsl2 wild-type (WT). Survival and growth curves The survival up 20 days or up to 3 months was recorded. A Kaplan-Meier survival curve was done to visualize the survival rate during the first 3 months as previously described. 31 To determine significant differences, the curves were analyzed with the Gehan-Breslow-Wilcoxon test that is better in determining significant difference in early time points. For growth curves, the weights at specific time points were recorded and plotted. Graph and student T-test done in Microsoft Excel (Redmond, WA). The number of animals is indicated in between parentheses in the figure. Euthanasia procedures Mice were placed inside a chamber and gradually filled with carbon dioxide for 5 minutes and then cervical dislocation was done to confirm death as approved by our IACUC, and according to the National Institutes of Health guidelines. Cell culture experiments All human dermal fibroblasts were isolated from skin biopsies from patients evaluated at the University of Miami. Written informed consents were obtained from all patients or their parents, including the use of the isolated primary fibroblasts to obtain and share research data. Dermal fibroblasts were obtained following the explant technique, as previously described. 31,36 Briefly, tissues were cut into small explants around 1x1 mm, and incubated at 37 o C and 5% CO 2 atmosphere, using complete Dulbecco’s Modified Eagle Medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, 11995-065), 20% FBS (Gen Clone, Genesee Scientific, 25–514), 100 U/mL of penicillin, 100 µg/mL of streptomycin (Corning, 30 − 002), and 200 µg/mL of Primocin (InvivoGen, ant-pm-05). The medium was changed every day until cells migrated from the explant and grew as a monolayer, and then every other day until the cells were cryopreserved as passage P2. Thawed fibroblasts for experiments were cultured in T75 flasks and used in a range from P4-P8 passages. All experiments were completed using Dulbecco’s Modified Eagle Medium (DMEM) high glucose, pyruvate, 10% FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Cells treatments : all cells were treated with 20 ng/mL TGF-β1 (Peprotech, 100-21-10UG), for 1 and 24 hours to check TGF-β pathway: p-SMAD2 (S465/467), SMAD2 and GAPDH. Cell lysates were obtained and analyzed by western bots. All cells were also treated with 20 ng/mL TGF-β1 and 20 ng/mL BMP2 (Sigma-Millipore, H4791-10UG) for 7 days, and media with the drug was changed every other day, to analyze their effect on the extracellular matrix proteins: ADAMTSL2, FBN1, fibronectin and GAPDH. Total lysates were obtained and analyzed by western bots, as previously described. 36 GD1-1 cells were treated with losartan at 3.3, 11, 33, 100 and 300 µM for 7 days; and media was changed every other day. Conditioned media were collected from third to sixth day (3 days) for analysis of TGF-β1 and extracellular protein secretion. Intracellular lysates and extracellular matrix lysates from losartan-treated cells were obtained for western blot analysis, always including ADAMTSL2, FBN1, fibronectin and GAPDH proteins. TGF-β1 quantification : TGF-β1 was quantified from conditioned media of dermal fibroblasts as previously described. 8,31,36 Briefly, 0.5 million cells were cultured in 12-well plates for a total of 7 days: first using complete DMEM high glucose, pyruvate, 10% FBS, 100 U/mL of penicillin, and 100 µg/mL of streptomycin. Media were changed every 2 days. After confluency (4 days later), cells were washed twice with PBS, and DMEM without FBS were added. Conditioned media were collected 72 hours later for further analysis. Human TGF-β1 DuoSet ELISA (R&D Systems, DY240), was performed according to manufacturer instructions. Protein lysates and samples Protein lysates were prepared using four different approaches depending on the experiment goal, as previously described. 36 1) Cell lysates (CL) for the TGF-β1 pathway proteins were prepared using 1x RIPA lysis buffer (Millipore-Sigma, R0278) containing 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), plus a 1x protease inhibitor mixture (C0mplete™ Protease Inhibitor Cocktail, Roche, 11697498001). RIPA was added directly to the cells, to avoid the addition of fresh medium containing FBS and a possible activation of the TGF-β1 pathway. Then the cells were scraped and incubated on ice for 15 minutes. Cell lysates were cleared at maximum speed (14000 rpm) for 5 minutes. 2) Intracellular lysates (IC) were prepared by adding RIPA buffer to trypsinized cells and incubated on ice for 15 minutes. Trypsin-EDTA (0.25%, Gibco 25200056) was added to the cells and incubated at 37 o C for 5 minutes. Trypsin was inhibited using complete medium DMEM (10% FBS) and cells were washed with PBS. Intracellular lysates were cleared at maximum speed (14000 rpm) for 5 minutes. Protein concentration was determined using a Bradford colorimetric assay kit (Bio-Rad Protein Assay Kit-II Bio-Rad, 500-0002). 3) Total lysates (Total) were prepared by adding 2x Laemmli buffer (Bio-Rad, 1610747) containing 10% β-mercaptoethanol (Millipore-Sigma, M3148-100ML), directly to the cells in well, previously washed with PBS. 4) ECM protein lysates were prepared using the Ammonium hydroxide (NH 4 OH) / Triton-X100 protocol. 66 Briefly, cells were washed with PBS and removed from the plate with 20 mM NH 4 OH, 0.05% Triton-X100 in PBS for 15 minutes at room temperature. The remaining ECM proteins attached to the plate (decellularized ECM) were washed with PBS and dissolve in 2x Laemmli buffer as 138.9 mM Tris-HCl, pH 6.8, 22.2% (v/v) glycerol, 2.2% LDS and 0,01% bromophenol blue containing 10% 2-mercaptoethanol. 5) Conditioned media were collected using different medium composition and different time points as specified in experiments. Western Blot Analysis WB were preformed following a regular protocol as previously described. 36 Ten to twenty µg of proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using the appropriate gel, and transferred to 0.22 µm PVDF membrane (Bio-Rad, 1620112). After blocking with 5% BSA in Tris Buffered Saline (TBS-T20, 25mM Tris, 140 mM Sodium Chloride, and 3.0 mM Potassium Chloride, and containing 0.1% Tween-20); the membranes were incubated with the corresponding primary antibody overnight at 4 o C ( Table S4 ). Primary antibodies were detected by horseradish peroxidase conjugated secondary antibody and the immunocomplexes were visualized by chemiluminescence (Super Signal™ West, Thermo-Fisher Scientific, 34577 and 34096). The membranes were stripped using Restore™ PLUS Western Blot Stripping Buffer (Thermo Scientific, 46430) at room temperature, for 15 minutes, extensively washed with TBS-T20, blocked with 5% BSA in TBS-T20, and reprobed with the corresponding antibodies following a similar protocol. Whole transcriptome sequencing (RNA-Seq) : RNA-Seq analysis was performed as previously reported. 36 Briefly, total RNA was extracted from primary human fibroblast, at P4. The cells were grown in DMEM 10% FBS, but the serum was removed overnight the day before the collection. The RNA was extracted using the RNeasy Mini Kit (Qiagen). A Bioanalyzer 2000 was used to measure the quality of RNA. All samples’ RNA integrity numbers (RIN) were above 9. Whole transcriptome sequencing (RNA-seq) was conducted at the Sequencing Core of John P. Hussman Institute of Human Genomics at the University of Miami using the TruSeq Stranded Total RNA Library Prep Kit from Illumina (San Diego, CA). Briefly, after ribosomal RNA (rRNA) was depleted, sequencing libraries were ligated with standard Illumina adaptors and subsequently sequenced on a Hiseq2000 sequencing system (100 bp single end reads, Illumina, San Diego, CA, USA). Trimming was performed, and sequence reads were aligned to the human transcriptome (GRCh38/hg38 assembly from the Genome Reference Consortium) and quantified using the STAR aligner. 67 All samples had uniquely aligned reads between 35,607,220 and 51,912,532. Read data was run through quality control metrics using MultiQC. 68 Statistical significance was first examined with three alternative differential expression calculators: edgeR, DESeq2, and Bayseq. 69–71 For visualization and further analysis iDEP software was used. 72 The program calculates CPM values from raw counts. To reduce false positive a minimum of 5 CPM per library was used. Data was transformed, normalized for clustering with rlog (transformation implemented in DESeq2). Of 60,656 genes from the data set, 11,147 genes passed the filter. A supervised Hierarchical and K-Means clustering with heatmap was generated to have a specific view of the expression of metalloprotease family of genes. Gene Set Enrichment Analysis (GSEA) database was used to identify all genes associated with the TGF-β signaling pathway, as well as genes associated with TGF-β1 stimulation, including reported up-regulated and down-regulated genes. 73,74 Statistics Data are expressed as mean ± SEM (standard error of the mean) and all graphs were performed in Microsoft Excel. The number of replicates for experiments are indicated in each figure and statistical differences were tested using the Student t-test, 1-way or 2-way Anova when comparing samples or a group with the corresponding control(s). p-values < 0.05 were considered statistically significant as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. Declarations Role of the funders : The funders of the present study had no role in study design, data collection, analysis, and interpretation, or during the writing of the manuscript. Author contributions AAM designed and conducted all cells experiments, acquired and analyzed data, and wrote the manuscript. VC designed and conducted animal experiments, acquired and analyzed data, and wrote and reviewed the manuscript. LP and SS recruited patients and reviews the manuscript. KW designed animal experiments, analyzed data and reviewed the manuscript. GW analyzed data and reviewed the manuscript. MT designed the research study, analyzed data, reviewed and approved the manuscript. Declaration of interests The authors or any immediate family members have no relevant financial or non-financial interests to disclose. All author affiliations have been declared. All funding sources for this study are listed in the acknowledgments section of the manuscript. The authors and our immediate family members, have no related patent applications or registrations to declare. Data availability The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. The datasets generated and/or analyzed during the current study are available in the ClinVar repository. Submission IDs for reported gene variants include: ADAMTSL2 c.182G> A as SUB15022370, ADAMTSL2 c.493G> A as SUB15022407, ADAMTSL2 c.542 T> C as SUB15022419, ADAMTSL2 c.707 C> T as SUB15022429 and FBN1 c.5284G> A as SUB15022437, FBN1 c.5183 C> T as SUB15022369 and FBN1 c.5243G> C as SUB15022471. RNASeq data are deposited at GEO with accession number GSE292600. Funding We are immensely grateful to the patients for their participation in this study. This study was supported by a gift from the Al Rashid Family. References Marzin, P. & Cormier-Daire, V. Geleophysic Dysplasia. 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1","display":"","copyAsset":false,"role":"figure","size":1204962,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLosartan potassium does not improve survival or growth in a GD1 animal model. (A)\u003c/strong\u003e Losartan treatment 38.25 mg/kg/day does not improve survival in Adamtsl2 mutant mice. Kaplan-Meier survival curve of mice with p.A165T allelic variant in ADAMTSL2 with and without Losartan treatment during the first 3 months. The curves were compared with Gehan-Breslow-Wilconox test to determine significance. A significant reduction in survival during the first 3 months was expected in \u003cem\u003eAdamtsl2\u003c/em\u003ep.A165T hemizygous (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001) mice vs. WT mice. The survival did not improve after Losartan treatment of \u003cem\u003eAdamtsl2\u003c/em\u003e p.A165T hemizygous. The treatment was administered in the drinking water of the dams at P18 and continued for 3 months postnatally in the drinking water of the mice. (N= number of animals) (WT \u003cem\u003eAdamtsl2\u003c/em\u003e= Black line, and p.A165T hemizygous = red line. No treatment (Continue line) and Losartan (discontinued dot line). \u003cstrong\u003e(B) \u003c/strong\u003eGrowth Curve male (top) and female (bottom). Average +/- SEM (*, \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/c259b0c31b50a6f7d8b9a564.png"},{"id":99222433,"identity":"52a57d44-156a-494e-9cf1-ebf36ac317b4","added_by":"auto","created_at":"2025-12-30 09:54:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2395141,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTGF-β1 pathway is not significantly dysregulated in patient fibroblasts\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e TGF-β1 was determined by ELISA, in conditioned media after 3 days culture in DMEM without FBS. Close and dashed lines in bars represent the active and the total TGF-β1, respectively. Averages and standard error of the mean (SEM) were calculated for 5 independent experiments. \u003cstrong\u003e(B)\u003c/strong\u003e Proteins related to TGF-β pathway were determined in protein lysates by WB: p-SMAD2 (S465/467), p-SMAD3 (S423/425), Smad4 and p-SMAD1/5/8 (S465/467). Representative images were selected from seven and four independent experiments for p-SMAD2 and others, respectively. Original uncropped blots are shown in Figure S2 \u003cstrong\u003e(C)\u003c/strong\u003eDensitometry analysis was completed using ImageJ software, for p-SMAD2/SMAD2, SMAD2/GAPDH, p-SMAD3/SMAD3, SMAD3/GAPDH, SMAD4/GAPDH, p-SMAD1/5/8 / SMAD1 (no signal for experiments) and SMAD1/GAPDH ratios of WBs in (B). \u003cstrong\u003e(D)\u003c/strong\u003e TGF-β pathway showed normal response after cells were stimulated with TGF-β1 for 24 hours and p-SMAD2 and SMAD2 were determined by WB. Original uncropped blots are shown in Figure S3. GAPDH was used as loading control for all WBs studies. Significance was calculated using a t-Student test and values were compared versus all values of CT fibroblasts. (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001). Bars and error bars represent the average and SEM for all values, respectively for four independent experiments.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/8436f748fe2d9a65c6e5f45e.png"},{"id":99222427,"identity":"d55b45c0-0449-45e6-b15c-ab32213dc820","added_by":"auto","created_at":"2025-12-30 09:54:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2078487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExogeneous TGF-β1 strongly induces up-regulation of key proteins of the ECM: ADAMTSL2, FBN1 and fibronectin\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003e Control and patient fibroblasts were treated with TGF-β1 (20 ng/mL) and BMP2 (20 ng/mL) for 7 days. Proteins related to TGF-β and BMP2 pathways were determined in Total lysates by WB. Pathway activation was measured by p-SMAD2 (S465/467) and p-SMAD1/5/8 (S465/467), respectively; in addition to the ECM: ADAMTSL2, FBN1 and fibronectin. Representative images were selected from four independent experiments. GAPDH was used as loading control. Results for CT and GD1 cells are separated from GD2 cells as they are from independent membranes. Original uncropped blots are shown in Figure S7.\u003cstrong\u003e (B)\u003c/strong\u003e Densitometry analysis was completed using ImageJ software and significance was calculated using a t-Student test and values were compared versus all values of untreated (UT) fibroblasts. (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, *** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.001 and **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001). Bars and error bars represent the average and SEM for all values, respectively for four independent experiments.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/2531821d14d48f13906372ca.png"},{"id":99222431,"identity":"0c5e767d-c09a-46d1-a065-c5ee9e58d261","added_by":"auto","created_at":"2025-12-30 09:54:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2098774,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLosartan does not affect TGF-β1 levels or ECM component in\u003c/strong\u003e \u003cstrong\u003epatient fibroblasts\u003c/strong\u003e. \u003cstrong\u003e(A)\u003c/strong\u003eCells were treated with increasing concentration of losartan for 7 days and conditioned media (CM) were collected from third day to the sixth day (3 days culture) and TGF-β1 was determined by ELISA. Close and dashed lines in bars represent the active and the total TGF-β1, respectively. Average and standard error of the mean (SEM) were calculated for 5 independent experiments. No significance was obtained by a t-Student test when values were compared versus CT or untreated cells.\u003cstrong\u003e \u003c/strong\u003eOriginal uncropped blots are shown in Figure S8\u003cstrong\u003e. (B)\u003c/strong\u003e Intracellular lysates (IC), ECM lysates as well as conditioned media (CM, 3 days) were used for WBs studies for ADAMTSL2, FBN1, fibronectin and Col1A1. GAPDH was used as loading control. Independent blot/membranes were used for IC, ECM and CM protein analysis. \u003cstrong\u003e(C)\u003c/strong\u003e Densitometry analysis was completed using ImageJ software, and all values were normalized by GAPDH. Averages and standard error of the mean (SEM) were calculated for 4 independent experiments. Significance was calculated using a t-Student test and values were compared versus all values of CT fibroblasts. (* \u003cem\u003ep\u003c/em\u003e\u0026lt;0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.01, and ***\u003cem\u003e p\u003c/em\u003e\u0026lt;0.001 and **** \u003cem\u003ep\u003c/em\u003e\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/01769e6d1dc43c4f206a4fd3.png"},{"id":99788337,"identity":"1bd67929-c243-4a6b-b17e-6f3154edd0ff","added_by":"auto","created_at":"2026-01-08 12:46:21","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10100283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/88ec4537-fd03-43cf-b203-0f4ab06d6813.pdf"},{"id":99222429,"identity":"feda2e5e-e5d5-4fbe-809a-e61415b68e80","added_by":"auto","created_at":"2025-12-30 09:54:29","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1533934,"visible":true,"origin":"","legend":"","description":"","filename":"TGF1Supplemental121625.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8301608/v1/fc74d4511907ff9e8f385a49.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Losartan shows limited benefit in preclinical models of Geleophysic dysplasia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGeleophysic dysplasia (GD) is a progressive genetic disorder presenting with short stature, short fingers and toes, joint contractures, distinctive facial features, and cardiopulmonary complications that contribute to poor prognosis. \u003csup\u003e1\u0026ndash;7\u003c/sup\u003e To date, three genes have been linked to GD: Geleophysic dysplasia type 1 (GD1, GPHYSD1, OMIM 231050) is an autosomal recessive form caused by mutations in \u003cem\u003eADAMTSL2\u003c/em\u003e (OMIM 612277). \u003csup\u003e8\u003c/sup\u003e GD type 2 (GD2, GPHYSD2, OMIM 614185) is autosomal dominantly inherited resulting from mutations in exons 41 or 42 of \u003cem\u003eFBN1\u003c/em\u003e (OMIM 134797). \u003csup\u003e9\u003c/sup\u003e GD type 3 (GD3, GPHYSD3, OMIM 614185) is also autosomal dominant and caused by variants in \u003cem\u003eLTBP3\u003c/em\u003e (OMIM 602090). \u003csup\u003e10\u003c/sup\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eADAMTSL2\u003c/em\u003e encodes a secreted matricellular glycoprotein within the ADAMTS (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA D\u003c/span\u003eisintegrin \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eA\u003c/span\u003end \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eM\u003c/span\u003eetalloprotease with \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eT\u003c/span\u003ehrombospondin motifs) superfamily, but it lacks the metalloprotease domain typical of this group. \u003csup\u003e11\u0026ndash;13\u003c/sup\u003e Although the function of ADAMTSL2 is not fully understood, emerging evidence points to its role in organizing fibrillin microfibrils \u003csup\u003e\u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and modulating the availability of Transforming Growth Factor Beta (TGF-β) via interaction with extracellular matrix (ECM) proteins such as FNB1 and LTBP1. \u003csup\u003e8,9,18\u0026ndash;21\u003c/sup\u003e Fibrillin-1, encoded by the \u003cem\u003eFBN1\u003c/em\u003e gene, is a large, calcium-binding ECM glycoprotein and a key structural component of microfibrils, with a regulatory role in the bioavailability of molecules like TGF-β. \u003csup\u003e20\u0026ndash;24\u003c/sup\u003e FBN1 mutations can cause Marfan syndrome, \u003csup\u003e25,26\u003c/sup\u003e but variants, in exons 41\u0026ndash;42, which encode the TGF-β-binding protein-like domain 5 (TB5), can result in GD2. \u003csup\u003e8,9,26\u003c/sup\u003e These mutations are thought to contribute to GD2 through disruption of TGF-β signaling. Latent Transforming Growth Factor Beta Binding Protein 3 (LTBP3) is an extracellular matrix protein that regulates the availability of TGF-β. \u003csup\u003e27,28\u003c/sup\u003e Variants in \u003cem\u003eLTBP3\u003c/em\u003e have been linked to genetic disorders with skeletal dysplasia, including GD3. \u003csup\u003e10,29,30\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eFour major mechanisms have been proposed in GD pathogenesis: First, mutations in \u003cem\u003eADAMTSL2\u003c/em\u003e, \u003cem\u003eFBN1\u003c/em\u003e and \u003cem\u003eLTBP3\u003c/em\u003e genes have been shown to impair protein secretion disrupting the ECM organization. \u003csup\u003e8,28,31\u0026ndash;33\u003c/sup\u003e Second, an increased TGF-β activity has been observed in cells from individuals with GD. \u003csup\u003e8\u0026ndash;10,15,16,18,34,35\u003c/sup\u003e Third, our recent findings implicate matrix metalloproteinases (MMPs), which are a family of enzymes responsible for ECM remodeling, as potential contributors to GD pathology. \u003csup\u003e36\u003c/sup\u003e Four, the mutations cause a reduction in the Wnt signaling pathway. \u003csup\u003e37\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA clear genotype-phenotype correlation in patients with GD has not yet been established. However, our recent work demonstrates that the clinical severity of GD1 correlates with the abundance of ADAMTSL2 in the extracellular matrix. \u003csup\u003e31\u003c/sup\u003e Using both cellular and murine models that replicate the genetic profile of a GD patient compound heterozygous for two \u003cem\u003eADAMTSL2\u003c/em\u003e variants, p.R61H and p.A165T, we observed the impaired secretion of ADAMTSL2 in patient-derived dermal fibroblasts and in HEK-293T overexpression systems.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Notably, the p.A165T variant resulted in a more pronounced secretion defect.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Mice homozygous or hemizygous for the p.A165T variant exhibited growth impairment, respiratory and cardiac dysfunction, and early mortality.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eLosartan, an angiotensin II type 1 receptor blocker, has been proposed to ameliorate Marfan syndrome pathology through inhibition of TGF-β signaling. \u003csup\u003e38\u003c/sup\u003e Losartan could hypothetically offer therapeutic benefit in GD by modulating TGF-β pathway. In this study, we evaluated the effects of losartan in both \u003cem\u003ein-vivo\u003c/em\u003e and \u003cem\u003ein-vitro\u003c/em\u003e models of GD1, using optimized dosing strategies in a mouse model carrying the p.A165T \u003cem\u003eADAMTSL2\u003c/em\u003e variant, as well as in patient-derived fibroblasts. Our goal was to determine whether losartan could improve survival or growth in the animal model or restore the ECM organization in patient cells.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eLosartan treatment does not improve the survival of Adamtsl2 mutant mice\u003c/h2\u003e \u003cp\u003eTo test the effects of losartan \u003cem\u003ein-vivo\u003c/em\u003e, we utilized our previously generated GD animal models. Mice that were hemizygous for the \u003cem\u003eAdamtsl2\u003c/em\u003e p.A165T missense variant were selected for treatment due to their phenotypic severity, especially their reduced survival and stunted growth. \u003csup\u003e31\u003c/sup\u003e Initially, we followed the treatment scheme and dosage previously used in mouse models of Marfan syndrome, consisting of prenatal and postnatal losartan treatments of 0.6 g/liter in the drinking water. \u003csup\u003e39\u003c/sup\u003e We found an increased lethality in the offspring of mice regardless of genotype with less than 40% survival in the first month (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). This treatment scheme was not continued because of the high toxicity in WT mice.\u003c/p\u003e \u003cp\u003eThe toxicity of losartan for the offspring during gestation is well documented; it can cross the placenta barrier, and it is secreted with the milk. For these reasons, losartan is a category D pregnancy risk medication and contraindicated in pregnant women.\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Such toxicity was not found in previous studies with the Marfan syndrome mouse models; a possible explanation could be variations in the water intake among mice strains. \u003csup\u003e45,46\u003c/sup\u003e An adult mouse depending on the strain can drink from 2.5 to 50 mL of water per day. \u003csup\u003e45,46\u003c/sup\u003e Adult C57BL6 female mice with 25 g of weight, the same strain of our GD mice, drink around 6.4 mL water per day. \u003csup\u003e45\u003c/sup\u003e Most of the toxicity studies in animals have been done on rats. The maximum dose in rats was calculated to be 10 to 20 mg/kg/day, and in humans 1 to 3 mg/kg/day. \u003csup\u003e40,47\u003c/sup\u003e In mice, previous studies have used losartan within 10 to 42.5 mg/kg/day. \u003csup\u003e40,48,49\u003c/sup\u003e Based on this information, we calculated that a dose 0.6 g/L in the drinking water provides 153 mg/kg/day of losartan in our animals, which is well above the estimated maximum dose. We proceeded to do a dose curve with our WT animals to determine the dose of losartan (from E18 to postnatal day 20) that could be tolerated without a significant effect in survival (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e) and found that 0.15 g/L in their drinking water provides a 38.25 mg/kg/day of losartan and has a minimal toxicity (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e). Therefore, we used this optimized dose to explore the potential therapeutic effect of losartan in the GD animal models.\u003c/p\u003e \u003cp\u003eOur previous data show that p.A165T hemizygous mice (A165T/-) present with a significant reduction in survival (~\u0026thinsp;40%) after 3 months. \u003csup\u003e31\u003c/sup\u003e Most of the lethality was observed in the first few days after birth. When we treated mice with the losartan dose of 38.5 mg/kg/day from E18, we did not find a significant improvement in survival in the p.A165T hemizygous offspring (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Weight was monitored on days 5.5, 15.5, 22.5, and 36.5. Hemizygous p.A165T animals showed significantly reduced weight, starting from 15.5 days of age in males and females, when compared to WT, regardless of the treatment status (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in treated p.A165T hemizygous males at 15.5, 22.5, and 36.5 days, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.07, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002 in treated females p.A165T hemizygous at 15.5, 22.5, and 36.5 days) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). These results indicate that using losartan in this treatment modality does not have a beneficial effect on the survival or growth of GD1 mouse models.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e At the end of all experiments, animals were euthanized using the carbon dioxide protocol and then cervical dislocation, as approved by our Institutional Animal Care and Use Committee (IACUC) and according to the National Institutes of Health guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFibroblasts from patients with GD1 and GD2\u003c/h3\u003e\n\u003cp\u003ePrevious studies, including ours, have shown that mutations in the \u003cem\u003eADAMTSL2\u003c/em\u003e and \u003cem\u003eFBN1\u003c/em\u003e genes impair the secretion of these proteins. \u003csup\u003e8,9,12,31\u003c/sup\u003e GD006 patient fibroblasts (GD1-2) were kindly provided by the Bristol Genetics Laboratory at North Bristol NHS Trust, Severn Pathology, Southmead Hospital, UK. To further investigate the cellular and molecular consequences of these variants, we analyzed primary human dermal fibroblasts obtained from patients with \u003cem\u003eADAMTSL2\u003c/em\u003e or \u003cem\u003eFBN1\u003c/em\u003e mutations. Fibroblasts from healthy individuals were used as controls. Individuals with LTBP3-related GD (GD3) were excluded from this study due to the rarity of this subtype, which accounts for less than 1% of the GD cases. \u003csup\u003e1\u003c/sup\u003e All fibroblasts were isolated using the explant technique, as previously described. \u003csup\u003e31,36\u003c/sup\u003e The gene variants identified in the fibroblast samples are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and were confirmed by sequencing of the \u003cem\u003eADAMTSL2\u003c/em\u003e and \u003cem\u003eFBN1\u003c/em\u003e genes. GD1 patients carried compound heterozygous mutations in the \u003cem\u003eADAMTSL2\u003c/em\u003e gene, while GD2 patients had heterozygous mutations in the \u003cem\u003eFBN1\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDetails of studied samples.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCT-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCT-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGD1-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGD1-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGD2-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGD2-2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGD2-3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDiagnosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGD1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGD2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGD2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePatient ID\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGD016E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGD017E\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGD001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGD006\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGD004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGD009\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGD012\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eMutations\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eADAMTSL2\u003c/b\u003e c.182G\u0026thinsp;\u0026gt;\u0026thinsp;A (\u003cb\u003ep.R61H)\u003c/b\u003e c.493G\u0026thinsp;\u0026gt;\u0026thinsp;A (\u003cb\u003ep.A165T)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eADAMTSL2\u003c/b\u003e c.542T\u0026thinsp;\u0026gt;\u0026thinsp;C (\u003cb\u003ep.V181A)\u003c/b\u003e c.707C\u0026thinsp;\u0026gt;\u0026thinsp;T (\u003cb\u003ep.P236L)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eFBN1\u003c/b\u003e c.5284G\u0026thinsp;\u0026gt;\u0026thinsp;A (\u003cb\u003ep.G1762S)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eFBN1\u003c/b\u003e c.5183C\u0026thinsp;\u0026gt;\u0026thinsp;T (\u003cb\u003ep.A1728V)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eFBN1\u003c/b\u003e c.5243G\u0026thinsp;\u0026gt;\u0026thinsp;C (\u003cb\u003ep.C1748S)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecompound heterozygous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ecompound heterozygous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eheterozygous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eheterozygous\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eheterozygous\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSex\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003efemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003emale\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge - years (months)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0 (1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0 (6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1 (11)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e28 (9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e11 (0)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eCT: Healthy control, GD1: Geleophysic Dysplasia type 1, GD2: Geleophysic Dysplasia type 2.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eGD fibroblasts do not show activation of TGF-β pathway\u003c/h3\u003e\n\u003cp\u003eTo assess TGF-β production, we used an ELISA assay under three different culture conditions using DMEM medium as the base: 10% FBS, 1% FBS, and serum-free (0% FBS). Although all conditions yielded similar trends, we chose the serum-free condition for subsequent analyses to eliminate potential cross-reactivity from bovine TGF-β and to evaluate TGF-β1 secretion without external stimulation. On average, the active form of TGF-β1 comprised of only about 1% of total TGF-β1 secreted from the cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The control cells secreted approximately 200 pg/mL over three days. In comparison, patient cells secreted an average of 114 pg/mL, indicating that fibroblasts from patients secreted significantly less amount of total TGF-β1 than controls.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next examined downstream TGF-β signaling by assessing SMAD2 and SMAD3 phosphorylation. Consistent with the low levels of active TGF-β, phosphorylated SMAD2 (S465/467) and SMAD3 (S423/425) were not detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC \u003cb\u003eand Figure S2)\u003c/b\u003e. Total SMAD2 was at similar levels between control and patient cells, while patient fibroblasts showed slightly reduced levels of total SMAD3 and SMAD4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Phosphorylated SMAD1/5/8 (S463/465) was also undetectable in all samples, and total SMAD1 levels did not differ between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTo get a deeper insight into TGF-β pathway on patient cells, we analyzed the fibroblast RNA-Seq data to profile the cell transcriptome and to identify active genes and pathways. We identified all genes associated with the TGF-β signaling pathway based on the Gene Set Enrichment Analysis database (GSEA, see methods). The transcript expression for all these selected genes was analyzed from the RNA-Seq data from control and patient-derived fibroblasts. The gene clustering did not reveal any significant dysregulation of the TGF-β pathway members between groups. Among the 56 genes analyzed, none showed a\u0026thinsp;\u0026ge;\u0026thinsp;2-fold change in expression between patient cells and controls (\u003cb\u003eFigure S4\u003c/b\u003e). Detailed transcript expressions for all genes are shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. In a similar way, we next examined genes previously reported to be upregulated following TGF-β1 stimulation (GSEA) and the RNA-Seq data for our cells was analyzed. The gene clustering did not indicate a consistent upregulation of these genes in patient samples (\u003cb\u003eFigure S5\u003c/b\u003e). Of the 116 genes evaluated, only one gene, \u003cem\u003eZIC4\u003c/em\u003e, was significantly upregulated in patient cells (\u003cb\u003eTable S2\u003c/b\u003e). Conversely, three genes, \u003cem\u003eCFAP298\u003c/em\u003e, \u003cem\u003eSLC8A1\u003c/em\u003e, and \u003cem\u003eJADE1\u003c/em\u003e, were significantly downregulated (\u0026ge;\u0026thinsp;2-fold), arguing against a generalized activation of TGF-β signaling (\u003cb\u003eTable S2\u003c/b\u003e). Finally, we also analyzed genes reported to be downregulated in response to TGF-β stimulation (GSEA). No clear pattern of downregulation emerged from gene clustering and shown in \u003cb\u003eFigure S6\u003c/b\u003e. Among the 176 genes assessed, 10 (\u003cem\u003eLAMA5\u003c/em\u003e, \u003cem\u003eGATA6\u003c/em\u003e, \u003cem\u003eTGM2\u003c/em\u003e, \u003cem\u003ePLAT\u003c/em\u003e, \u003cem\u003eSSX2IP\u003c/em\u003e, \u003cem\u003eSYNE2\u003c/em\u003e, \u003cem\u003eC4B\u003c/em\u003e, \u003cem\u003eEZR\u003c/em\u003e, \u003cem\u003eMET\u003c/em\u003e, and \u003cem\u003eALCAM\u003c/em\u003e) were significantly downregulated in patient cells, while one gene, \u003cem\u003eMTUS1\u003c/em\u003e, was significantly upregulated (\u003cb\u003eTable S3\u003c/b\u003e). Collectively, these analyses do not support the hypothesis of TGF-β pathway activation in the patient-derived fibroblasts.\u003c/p\u003e \u003cp\u003eTo evaluate whether patient fibroblasts retained the ability to respond to TGF-β, we treated both control and patient cells with exogenous TGF-β1 for 1 and 24 hours, then assessed SMAD2 phosphorylation at the S465/467 site, a key event in TGF-β signaling. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD \u003cb\u003eand Figure S3\u003c/b\u003e, all patient cells responded robustly to TGF-β after 1 hour of stimulation, showing comparable levels of SMAD2 phosphorylation to controls. Similar results were observed after 24 hours (data not shown), indicating that TGF-β signaling pathway remains intact in patients` fibroblasts despite their reduced basal secretion of TGF-β1 or phosphorylation status of SMAD2 or SMAD3.\u003c/p\u003e\n\u003ch3\u003eStimulation of TGF-β signaling increases ADAMTSL2, FBN1 and fibronectin in fibroblasts\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of the TGF-β signaling pathway in the regulation of GD-associated proteins, we treated control and patient-derived fibroblasts with TGF-β1 or BMP2 for seven days and then assessed the levels of ADAMTSL2, FBN1, and fibronectin. To check the pathway activation, we measured SMAD2 phosphorylation in response to TGF-β1 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003eand Figure S7\u003c/b\u003e). TGF-β1 not only induced phosphorylation of SMAD2 but also the phosphorylation of Smad1/5/8, indicating the activation of both the canonical and non-canonical pathways, as previously reported. \u003csup\u003e50,51\u003c/sup\u003e In contrast, BMP2 selectively induced phosphorylation of Smad1/5/8, consistent with its known signaling pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTGF-β1 treatment led to a marked upregulation of ADAMTSL2 expression in all patient fibroblasts and in one control line, with increases observed both intracellularly and extracellularly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). BMP2 also increased ADAMTSL2 expression, albeit to a lesser extent and only in a subset of the cells tested. Similarly, TGF-β1 increased FBN1 expression in both patient and control fibroblasts, while BMP2 elicited a response only in control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Notably, the fold increase in FBN1 expression was greater in the patient fibroblasts, likely due to their lower baseline levels, as we previously described. \u003csup\u003e31,36\u003c/sup\u003e Fibronectin levels were elevated following TGF-β1 treatment in both patient and control fibroblasts, whereas BMP2 had no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). All these results highlight the key role of TGF-β pathway in the ECM organization and suggest that uncontrolled inhibition of the pathway might lead to additional ECM dysfunction.\u003c/p\u003e\n\u003ch3\u003eLosartan does not affect TGF-β signaling or ECM protein incorporation in patient fibroblasts\u003c/h3\u003e\n\u003cp\u003eTo investigate the effect of losartan on TGF-β1 signaling, patient-derived fibroblasts were treated with varying concentrations of the drug. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA \u003cb\u003eand Figure S8\u003c/b\u003e, losartan did not significantly alter total TGF-β1 levels in the conditioned media under 10% FBS conditions when compared to untreated controls. A slight increase in active TGF-β1 was observed at certain concentrations; however, these values remained below the detection limit of the ELISA and were therefore considered unreliable. At low concentrations, losartan did not affect the total TGF-β1 secretion in patient fibroblasts. Interestingly, treatment with losartan at 300 \u0026micro;M, resulted in a slight but significant increase in total TGF-β1 secretion in patient cells. Despite this increase, no differences in SMAD2 phosphorylation were observed upon losartan treatment, as demonstrated by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), suggesting that downstream TGF-β signaling remained unaltered.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with our previous findings, the ADAMTSL2 protein was predominantly detected in the intracellular (IC) compartment (bands-1 and band-2, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) with limited detection in the ECM fraction (bands-3 and band-4), relative to IC levels. \u003csup\u003e31,36\u003c/sup\u003e GAPDH and fibronectin served as controls for IC and ECM fractions, respectively. \u003cem\u003eADAMTSL2\u003c/em\u003e mutations did not affect its intracellular expression, and no significant differences were observed between control and patient fibroblasts. However, secretion of ADAMTSL2 into the conditioned media and its incorporation into the ECM were significantly reduced in patient fibroblasts. Treatment with losartan, even at increasing concentrations, did not alter ADAMTSL2 intracellular expression, secretion, or ECM incorporation (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn contrast to ADAMTSL2, FBN1 was primarily localized to the ECM, with minimal intracellular localization. \u003csup\u003e31,36\u003c/sup\u003e Notably, the intracellular form of FBN1 exhibited a slightly higher molecular weight compared to its secreted form (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). As previously reported, ADAMTSL2 mutations led to reduced intracellular FBN1 levels in patient fibroblasts relative to controls (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Although secretion of FBN1 was modestly affected, differences were not statistically significant. The most pronounced alteration was a substantial reduction in ECM-incorporated FBN1 in patient cells. Losartan treatment did not modify FBN1 intracellular levels, secretion into the conditioned media, or incorporation into the ECM.\u003c/p\u003e \u003cp\u003eWe further examined the expression and distribution of two additional ECM components, fibronectin and collagen type-I alpha 1 (Col1A1). Fibronectin was primarily detected in the ECM with limited intracellular presence, whereas Col1A1 was largely retained intracellularly with poor ECM incorporation. \u003csup\u003e31,36\u003c/sup\u003e In patient fibroblasts, fibronectin expression and secretion into the conditioned media were comparable to controls; however, ECM incorporation was markedly reduced. Col1A1 showed mildly increased intracellular expression in patient fibroblasts, but secretion levels were unchanged. Like fibronectin, Col1A1 incorporation into the ECM was significantly impaired in the patient cells. Importantly, losartan treatment had no observable effect on the intracellular expression, secretion, or ECM incorporation of either protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003ePrevious studies have suggested that dysregulation of TGF-β signaling is the underlying mechanism in GD. Fibroblasts derived from patients with mutations in \u003cem\u003eADAMTSL2\u003c/em\u003e and \u003cem\u003eFBN1\u003c/em\u003e genes have consistently shown altered TGF-β signaling. \u003csup\u003e1,8,9,20,21,23,24\u003c/sup\u003e Although GD-associated mutations for the \u003cem\u003eLTBP3\u003c/em\u003e gene similarly disrupt the microfibrillar network, it does not appear to increase TGF-β signaling in the dermal fibroblast model described for GD3.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e Nevertheless, in a different cellular model for adipogenesis, the loss of LTBP3 resulted in a reduced adipogenesis via an increase in TGF beta signaling. \u003csup\u003e52\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the present study, our \u003cem\u003eADAMTSL2\u003c/em\u003e p.A165T mouse model displayed severe growth impairment and high perinatal mortality, yet losartan treatment failed to improve either outcome. Unexpectedly, we observed no evidence of TGF-β pathway activation in dermal fibroblasts derived from GD patients. Compared to control fibroblasts, patient cells secreted lower levels of TGF-β1 and lacked detectable SMAD2/3 phosphorylation. Treatment with active TGF-β induced phosphorylation of SMAD2 without significant differences between control and patient cells, suggesting that the signaling machinery remain intact. TGF-β1 and ADAMTSL2 are proposed to be involved in a bidirectional regulatory loop. \u003csup\u003e15,53\u003c/sup\u003e ADAMTSL2 inhibits TGF-β signaling by binding to LTBP1 within the microfibrillar network, thereby reducing the bioavailability of active TGF-β. \u003csup\u003e15,53\u003c/sup\u003e Conversely, TGF-β1 signaling can induce the expression of ADAMTSL2, suggesting a feedback mechanism wherein increased TGF-β activity promotes ADAMTSL2 expression to attenuate further signaling. \u003csup\u003e18,54\u003c/sup\u003e In this study, treatment with TGF-β1 and BMP2 led to increased expression of both ADAMTSL2 and FBN1 in all primary fibroblast. This suggests that upregulation of these ECM components by TGF-β1 is a generalizable mechanism in dermal fibroblasts, independent of the genotype.\u003c/p\u003e \u003cp\u003eOur findings suggest that an increase in TGF-β signaling is not the primary driver of disease pathology. In fact, there is evidence from other studies suggesting that an increased TGF-β signaling is not consistently observed across all models of GD. \u003csup\u003e15,31,37,55\u003c/sup\u003e For example, in \u003cem\u003eADAMTSL2\u003c/em\u003e-deficient myoblasts no increase in TGF-β activity was detected.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e In \u003cem\u003eAdamtsl2\u003c/em\u003e knockout mice, TGF-β signaling was not elevated at birth and pan-TGF-β neutralizing antibody failed to rescue the phenotype. \u003csup\u003e15\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA similar mechanism involving dysregulated TGF-β signaling has been proposed for Marfan syndrome, which is caused FBN1 mutations. \u003csup\u003e56\u0026ndash;58\u003c/sup\u003e Marfan syndrome patients present a phenotype that is notably opposite to that of GD, characterized by tall stature and long extremities. This striking contrast raises the question of how mutations in the same gene and potentially overlapping molecular pathways can lead to such divergent clinical manifestations. Numerous studies have evaluated losartan\u0026rsquo;s efficacy in Marfan syndrome animal models and patient cohorts,\u003csup\u003e59\u0026ndash;65\u003c/sup\u003e yet no investigation has examined its effects in GD patients or animal models. Notably, losartan was reported in 2019 to improve lysosomal inclusions and microfibril deposition in GD2 primary fibroblast in vitro. Motivated by this finding, and by recurrent inquiries from patients and clinicians about losartan as a potential therapy for GD, we investigated its efficacy in our GD models. In our study, losartan conferred no therapeutic benefit in GD1 mice and failed to improve ECM organization in patient primary fibroblasts, suggesting that GD and Marfan syndrome do not share a common pathogenic mechanism.\u003c/p\u003e \u003cp\u003eOur findings challenge the assumption that TGF-β dysregulation is a universal feature of GD and suggest that TGF-β-targeted therapies are not effective in GD. Further work is needed to elucidate the precise molecular pathways involved and to identify more targeted therapeutic strategies.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cp\u003e \u003cstrong\u003eStudy approval\u003c/strong\u003e \u003cp\u003e All human and animal studies were reviewed and approved at the University of Miami and by the Institutional Review Board (IRB) and by the Institutional Animal Care and Use Committee (AICUC), respectively, and according to the National Institutes of Health guidelines (NIH). All patients were evaluated at the University of Miami or by collaborators. Written informed consents for the testing and sharing of the research data were obtained from all patients or their parents, including the use of the isolated primary fibroblasts. All animal studies and experiments were performed following all relevant guidelines and regulations; and all reports were in accordance with ARRIVE guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eLosartan Potassium\u003c/strong\u003e \u003cp\u003eLosartan was obtained from Tokyo Chemical Industry, Tokyo Japan. Diluted in the drinking water at 0.6, 0.3, 0.15, and 0.075 g/L to provide 153, 76.5, 38.25, and 19.125 mg/kg/day respectively.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eTransgenic mice\u003c/strong\u003e \u003cp\u003efor the generation of \u003cem\u003eAdamtsl2\u003c/em\u003e p.A165T missense variant, we developed a knock-in mouse model carrying the variant by conventional embryonic stem cell-mediated knock-in technology using the service of Taconic-Cyagen Model generation Alliance (Germantown, NY), as previously described.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e In summary, a targeted C57BL/6J ES cell clone was injected into C57BL/6 albino embryos, Germline transmission was further confirmed after further breading with C57BL6 females. \u003cem\u003eAdamtsl2\u003c/em\u003e p.A165T heterozygous mice were crossed between them to obtain homozygous animals or with heterozygous mice carrying a knock-out allele of \u003cem\u003eAdamtsl2\u003c/em\u003e\u003csup\u003e14\u003c/sup\u003e, to obtain hemizygous mice. Animals were genotyped as previously described and housed under a 12-hour light-dark cycle at 20\u0026ndash;23\u0026deg;C in specific pathogen-free facilities and supplied with food and water ad libitum. All studies and experiments were performed in accordance with relevant guidelines and regulations. In addition, all reports were in accordance with ARRIVE guidelines. All litters, excluding the first one, from different types of matings, were included in these studies until at least 10 animals per group was reached. Dams with high litter mortality caused by others factor like complications during birth were removed from the study. \u003cem\u003eAdamtsl2\u003c/em\u003e p.A165T hemizygous mice were compared with \u003cem\u003eAdamtsl2\u003c/em\u003e wild-type (WT).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eSurvival and growth curves\u003c/strong\u003e \u003cp\u003eThe survival up 20 days or up to 3 months was recorded. A Kaplan-Meier survival curve was done to visualize the survival rate during the first 3 months as previously described.\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e To determine significant differences, the curves were analyzed with the Gehan-Breslow-Wilcoxon test that is better in determining significant difference in early time points. For growth curves, the weights at specific time points were recorded and plotted. Graph and student T-test done in Microsoft Excel (Redmond, WA). The number of animals is indicated in between parentheses in the figure.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eEuthanasia procedures\u003c/strong\u003e \u003cp\u003eMice were placed inside a chamber and gradually filled with carbon dioxide for 5 minutes and then cervical dislocation was done to confirm death as approved by our IACUC, and according to the National Institutes of Health guidelines.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell culture experiments\u003c/strong\u003e \u003cp\u003eAll human dermal fibroblasts were isolated from skin biopsies from patients evaluated at the University of Miami. Written informed consents were obtained from all patients or their parents, including the use of the isolated primary fibroblasts to obtain and share research data. Dermal fibroblasts were obtained following the explant technique, as previously described. \u003csup\u003e31,36\u003c/sup\u003e Briefly, tissues were cut into small explants around 1x1 mm, and incubated at 37\u003csup\u003eo\u003c/sup\u003eC and 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere, using complete Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) high glucose, pyruvate (Thermo Fisher Scientific, 11995-065), 20% FBS (Gen Clone, Genesee Scientific, 25\u0026ndash;514), 100 U/mL of penicillin, 100 \u0026micro;g/mL of streptomycin (Corning, 30\u0026thinsp;\u0026minus;\u0026thinsp;002), and 200 \u0026micro;g/mL of Primocin (InvivoGen, ant-pm-05). The medium was changed every day until cells migrated from the explant and grew as a monolayer, and then every other day until the cells were cryopreserved as passage P2. Thawed fibroblasts for experiments were cultured in T75 flasks and used in a range from P4-P8 passages. All experiments were completed using Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) high glucose, pyruvate, 10% FBS, 100 U/mL of penicillin, and 100 \u0026micro;g/mL of streptomycin.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCells treatments\u003c/b\u003e: all cells were treated with 20 ng/mL TGF-β1 (Peprotech, 100-21-10UG), for 1 and 24 hours to check TGF-β pathway: p-SMAD2 (S465/467), SMAD2 and GAPDH. Cell lysates were obtained and analyzed by western bots. All cells were also treated with 20 ng/mL TGF-β1 and 20 ng/mL BMP2 (Sigma-Millipore, H4791-10UG) for 7 days, and media with the drug was changed every other day, to analyze their effect on the extracellular matrix proteins: ADAMTSL2, FBN1, fibronectin and GAPDH. Total lysates were obtained and analyzed by western bots, as previously described.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e GD1-1 cells were treated with losartan at 3.3, 11, 33, 100 and 300 \u0026micro;M for 7 days; and media was changed every other day. Conditioned media were collected from third to sixth day (3 days) for analysis of TGF-β1 and extracellular protein secretion. Intracellular lysates and extracellular matrix lysates from losartan-treated cells were obtained for western blot analysis, always including ADAMTSL2, FBN1, fibronectin and GAPDH proteins.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTGF-β1 quantification\u003c/b\u003e: TGF-β1 was quantified from conditioned media of dermal fibroblasts as previously described. \u003csup\u003e8,31,36\u003c/sup\u003e Briefly, 0.5\u0026nbsp;million cells were cultured in 12-well plates for a total of 7 days: first using complete DMEM high glucose, pyruvate, 10% FBS, 100 U/mL of penicillin, and 100 \u0026micro;g/mL of streptomycin. Media were changed every 2 days. After confluency (4 days later), cells were washed twice with PBS, and DMEM without FBS were added. Conditioned media were collected 72 hours later for further analysis. Human TGF-β1 DuoSet ELISA (R\u0026amp;D Systems, DY240), was performed according to manufacturer instructions.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eProtein lysates and samples\u003c/strong\u003e \u003cp\u003eProtein lysates were prepared using four different approaches depending on the experiment goal, as previously described.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e1) \u003cb\u003eCell lysates\u003c/b\u003e (CL) for the TGF-β1 pathway proteins were prepared using 1x RIPA lysis buffer (Millipore-Sigma, R0278) containing 50 mM Tris-HCl, pH 8.0, with 150 mM sodium chloride, 1.0% Igepal CA-630 (NP-40), 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate (SDS), plus a 1x protease inhibitor mixture (C0mplete\u0026trade; Protease Inhibitor Cocktail, Roche, 11697498001). RIPA was added directly to the cells, to avoid the addition of fresh medium containing FBS and a possible activation of the TGF-β1 pathway. Then the cells were scraped and incubated on ice for 15 minutes. Cell lysates were cleared at maximum speed (14000 rpm) for 5 minutes.\u003c/p\u003e \u003cp\u003e2) \u003cb\u003eIntracellular lysates\u003c/b\u003e (IC) were prepared by adding RIPA buffer to trypsinized cells and incubated on ice for 15 minutes. Trypsin-EDTA (0.25%, Gibco 25200056) was added to the cells and incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 5 minutes. Trypsin was inhibited using complete medium DMEM (10% FBS) and cells were washed with PBS. Intracellular lysates were cleared at maximum speed (14000 rpm) for 5 minutes.\u003c/p\u003e \u003cp\u003eProtein concentration was determined using a Bradford colorimetric assay kit (Bio-Rad Protein Assay Kit-II Bio-Rad, 500-0002).\u003c/p\u003e \u003cp\u003e3) \u003cb\u003eTotal lysates\u003c/b\u003e (Total) were prepared by adding 2x Laemmli buffer (Bio-Rad, 1610747) containing 10% β-mercaptoethanol (Millipore-Sigma, M3148-100ML), directly to the cells in well, previously washed with PBS.\u003c/p\u003e \u003cp\u003e4) \u003cb\u003eECM protein lysates\u003c/b\u003e were prepared using the Ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH) / Triton-X100 protocol. \u003csup\u003e66\u003c/sup\u003e Briefly, cells were washed with PBS and removed from the plate with 20 mM NH\u003csub\u003e4\u003c/sub\u003eOH, 0.05% Triton-X100 in PBS for 15 minutes at room temperature. The remaining ECM proteins attached to the plate (decellularized ECM) were washed with PBS and dissolve in 2x Laemmli buffer as 138.9 mM Tris-HCl, pH 6.8, 22.2% (v/v) glycerol, 2.2% LDS and 0,01% bromophenol blue containing 10% 2-mercaptoethanol.\u003c/p\u003e \u003cp\u003e5) \u003cb\u003eConditioned media\u003c/b\u003e were collected using different medium composition and different time points as specified in experiments.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eWestern Blot Analysis\u003c/strong\u003e \u003cp\u003eWB were preformed following a regular protocol as previously described. \u003csup\u003e36\u003c/sup\u003e Ten to twenty \u0026micro;g of proteins were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), using the appropriate gel, and transferred to 0.22 \u0026micro;m PVDF membrane (Bio-Rad, 1620112). After blocking with 5% BSA in Tris Buffered Saline (TBS-T20, 25mM Tris, 140 mM Sodium Chloride, and 3.0 mM Potassium Chloride, and containing 0.1% Tween-20); the membranes were incubated with the corresponding primary antibody overnight at 4\u003csup\u003eo\u003c/sup\u003eC (\u003cb\u003eTable S4\u003c/b\u003e). Primary antibodies were detected by horseradish peroxidase conjugated secondary antibody and the immunocomplexes were visualized by chemiluminescence (Super Signal\u0026trade; West, Thermo-Fisher Scientific, 34577 and 34096). The membranes were stripped using Restore\u0026trade; PLUS Western Blot Stripping Buffer (Thermo Scientific, 46430) at room temperature, for 15 minutes, extensively washed with TBS-T20, blocked with 5% BSA in TBS-T20, and reprobed with the corresponding antibodies following a similar protocol.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWhole transcriptome sequencing (RNA-Seq)\u003c/b\u003e: RNA-Seq analysis was performed as previously reported. \u003csup\u003e36\u003c/sup\u003e Briefly, total RNA was extracted from primary human fibroblast, at P4. The cells were grown in DMEM 10% FBS, but the serum was removed overnight the day before the collection. The RNA was extracted using the RNeasy Mini Kit (Qiagen). A Bioanalyzer 2000 was used to measure the quality of RNA. All samples\u0026rsquo; RNA integrity numbers (RIN) were above 9. Whole transcriptome sequencing (RNA-seq) was conducted at the Sequencing Core of John P. Hussman Institute of Human Genomics at the University of Miami using the TruSeq Stranded Total RNA Library Prep Kit from Illumina (San Diego, CA). Briefly, after ribosomal RNA (rRNA) was depleted, sequencing libraries were ligated with standard Illumina adaptors and subsequently sequenced on a Hiseq2000 sequencing system (100 bp single end reads, Illumina, San Diego, CA, USA). Trimming was performed, and sequence reads were aligned to the human transcriptome (GRCh38/hg38 assembly from the Genome Reference Consortium) and quantified using the STAR aligner. \u003csup\u003e67\u003c/sup\u003e All samples had uniquely aligned reads between 35,607,220 and 51,912,532. Read data was run through quality control metrics using MultiQC. \u003csup\u003e68\u003c/sup\u003e Statistical significance was first examined with three alternative differential expression calculators: edgeR, DESeq2, and Bayseq.\u0026nbsp;\u003csup\u003e69\u0026ndash;71\u003c/sup\u003e For visualization and further analysis iDEP software was used. \u003csup\u003e72\u003c/sup\u003e The program calculates CPM values from raw counts. To reduce false positive a minimum of 5 CPM per library was used. Data was transformed, normalized for clustering with rlog (transformation implemented in DESeq2). Of 60,656 genes from the data set, 11,147 genes passed the filter. A supervised Hierarchical and K-Means clustering with heatmap was generated to have a specific view of the expression of metalloprotease family of genes.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eGene Set Enrichment Analysis (GSEA)\u003c/strong\u003e \u003cp\u003edatabase was used to identify all genes associated with the TGF-β signaling pathway, as well as genes associated with TGF-β1 stimulation, including reported up-regulated and down-regulated genes. \u003csup\u003e73,74\u003c/sup\u003e\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eStatistics\u003c/strong\u003e \u003cp\u003eData are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean) and all graphs were performed in Microsoft Excel. The number of replicates for experiments are indicated in each figure and statistical differences were tested using the Student t-test, 1-way or 2-way Anova when comparing samples or a group with the corresponding control(s). p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant as * p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ** p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, *** p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 and **** p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eRole of the funders\u003c/strong\u003e: The funders of the present study had no role in study design, data collection, analysis, and interpretation, or during the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAAM designed and conducted all cells experiments, acquired and analyzed data, and wrote the manuscript. VC designed and conducted animal experiments, acquired and analyzed data, and wrote and reviewed the manuscript. LP and SS recruited patients and reviews the manuscript. \u0026nbsp;KW designed animal experiments, analyzed data and reviewed the manuscript. GW analyzed data and reviewed the manuscript. MT designed the research study, analyzed data, reviewed and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDeclaration of interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors or any immediate family members have no relevant financial or non-financial interests to disclose. All author affiliations have been declared. All funding sources for this study are listed in the acknowledgments section of the manuscript. The authors and our immediate family members, have no related patent applications or registrations to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files. Should any raw data files be needed in another format they are available from the corresponding author upon reasonable request. The datasets generated and/or analyzed during the current study are available in the ClinVar repository. Submission IDs for reported gene variants include: \u003cem\u003eADAMTSL2\u003c/em\u003e c.182G\u0026gt; A as SUB15022370, \u003cem\u003eADAMTSL2\u003c/em\u003e c.493G\u0026gt; A as SUB15022407, \u003cem\u003eADAMTSL2\u003c/em\u003e c.542 T\u0026gt; C as SUB15022419, \u003cem\u003eADAMTSL2\u003c/em\u003e c.707 C\u0026gt; T as SUB15022429 and \u003cem\u003eFBN1\u003c/em\u003e c.5284G\u0026gt; A as SUB15022437, \u003cem\u003eFBN1\u0026nbsp;\u003c/em\u003ec.5183 C\u0026gt; T as SUB15022369 and \u003cem\u003eFBN1\u003c/em\u003e c.5243G\u0026gt; C as SUB15022471. RNASeq data are deposited at GEO with accession number GSE292600.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are immensely grateful to the patients for their participation in this study. This study was supported by a gift from the Al Rashid Family.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMarzin, P. \u0026amp; Cormier-Daire, V. Geleophysic Dysplasia. In: Adam MP, Feldman J, Mirzaa GM, Pagon RA, Wallace SE, Bean LJH, Gripp KW, Amemiya A, editors. \u003cem\u003eGeneReviews\u0026reg;\u003c/em\u003e [Internet]. Seattle (WA): University of Washington, Seattle; \u0026ndash;2024. (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpranger, J. W., Gilbert, E. F., Tuffli, G. A., Rossiter, F. P. \u0026amp; Opitz, J. M. Geleophysic dwarfism\u0026mdash;a focal mucopolysaccharidosis? \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 97\u0026ndash;98 (1971).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpranger, J., Gilbert, E. F., Arya, S., Hoganson, G. M. I. \u0026amp; Opitz, J. M. Geleophysic dysplasia. \u003cem\u003eAm. J. Med. 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PGC-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes. \u003cem\u003eNat. Genet.\u003c/em\u003e \u003cb\u003e34\u003c/b\u003e (3), 267\u0026ndash;273 (2003).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Geleophysic dysplasia, ADAMTSL2, FBN1, TGF-β1, losartan, extracellular matrix","lastPublishedDoi":"10.21203/rs.3.rs-8301608/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8301608/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGeleophysic dysplasia (GD) is a rare genetic disorder characterized by short stature, joint contractures, and cardiopulmonary complications, with early mortality, and linked to mutations in ADAMTSL2 (GD1), FBN1 (GD2), or LTBP3 (GD3) genes. These mutations are hypothesized to disrupt extracellular matrix (ECM) organization and enhance transforming growth factor beta (TGF-β) signaling. Losartan, an angiotensin II receptor blocker, has been proposed to mitigate TGF-β-mediated pathologies. In this study we tested the efficacy of losartan as a therapeutic drug for GD. We evaluated losartan's therapeutic potential using ADAMTSL2 p.A165T mutant mice and patient-derived fibroblasts. Survival, growth, TGF-β signaling, and ECM protein expression were assessed. Losartan did not improve survival or growth in mutant mice. Patient fibroblasts exhibited reduced basal TGF-β1 secretion and SMAD phosphorylation without transcriptomic evidence of pathway activation. Losartan treatment failed to modulate TGF-β signaling or ECM protein incorporation. These results suggest limited benefits of losartan in GD and challenge the notion of TGF-β dysregulation in GD pathogenesis, indicating a need for alternative targeted therapies.\u003c/p\u003e","manuscriptTitle":"Losartan shows limited benefit in preclinical models of Geleophysic dysplasia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-30 09:53:20","doi":"10.21203/rs.3.rs-8301608/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-09T06:55:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-08T23:50:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-01T02:32:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300661248007860380375009925805921953917","date":"2026-03-25T10:30:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75160353923071895647324943501550700235","date":"2026-03-21T22:23:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-02T13:15:46+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-15T09:24:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"257975497866668165050109453742707065181","date":"2025-12-26T21:34:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-25T09:01:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-25T08:51:07+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-23T12:50:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-19T17:58:57+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-19T17:52:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d95f563-03ec-4cfc-82dc-a741af5fa332","owner":[],"postedDate":"December 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":60296310,"name":"Biological sciences/Cell biology"},{"id":60296311,"name":"Health sciences/Diseases"},{"id":60296312,"name":"Biological sciences/Genetics"},{"id":60296313,"name":"Health sciences/Medical research"},{"id":60296314,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-12T07:23:24+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-30 09:53:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8301608","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8301608","identity":"rs-8301608","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}