{"paper_id":"0c5fc945-6f49-4e96-9b2a-4e2823c07711","body_text":"1 \n \n \nAngiotensin II Induces Abdominal Aortic Branch Aneurysms  \nin Fibrillin-1C1041G/+ Mice \n \n \nMichael K. Franklin,1 Deborah A. Howatt,1 Jessica J. Moorleghen,1  \nMary B. Sheppard,1-4 Hisashi Sawada,1-3 Hong S. Lu,1-3 Alan Daugherty1-3 \n \n1 Saha Cardiovascular Research Center \n2 Saha Aortic Center \n3 Department of Physiology, \n4 Department of Family and Community Medicine \nUniversity of Kentucky, Lexington, KY \n \n \nRunning title – Aortic Branch Aneurysms \n \nCorresponding authors: \nAlan Daugherty \nSaha CVRC, BBSRB, Room 243 \nUniversity of Kentucky \n741 S Limestone \nLexington KY 40356-0509 \ne-mail – alan.daugherty@uky.edu\n \n \nHong S. Lu \nSaha CVRC, BBSRB, Room 249 \nUniversity of Kentucky \n741 S Limestone \nLexington KY 40356-0509 \ne-mail – Hong.Lu@uky.edu\n \n \n \nAbbreviations \nAngiotensin II   AngII \nFibrillin-1    Fbn1 \nMicro Computed Tomography  microCT \nNorepinephrine   NE   \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n2 \n \n \nHIGHLIGHTS \n• Aortic branch aneurysms have recently been demonstrated to be prominent in \nMarfan patients but have not been reported in Marfan mouse models. \n• Angiotensin II infusion into fibrillin-1C1041G/+ mice promoted development of \nprominent aortic pathologies, including enhanced aneurysm and rupture in both \nthe thoracic and abdominal regions. \n• Angiotensin II infusion into fibrillin-1C1041G/+ mice promoted development of \npronounced aneurysms at the aortic branches of the celiac and superior \nmesenteric arteries.  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n3 \n \nABSTRACT \nBackground: \nMice harboring a missense variant (C1041G) of fibrillin-1 (Fbn1) have been used \nextensively for aortopathy research, but do not mimic all facets of the human disease. \nThe role of increased angiotensin II (AngII) or blood pressure in determining the arterial \nphenotype of these mice remains incompletely defined. The purpose of this study was \nto define whether AngII, directly or via increased blood pressure, promoted aortic \ndisease in the proximal thoracic aorta and beyond.  \nMethods: \nFbn1\n+/+ and Fbn1C1041G/+ littermates were infused with either AngII or \nnorepinephrine (NE) via subcutaneously implanted osmotic pumps. Aortic dimensions \nwere determined using in situ imaging. Micro Computed tomography (microCT) was \nused to determine the localization of aortic pathologies.  \nResults: \nAngII infusion dramatically augmented aortopathy in Fbn1\nC1041G/+ mice. Aortic \ndissection was visible within 3 days of AngII infusion. Over 50% of male Fbn1C1041G/+ \nmice died during AngII infusion, primarily due to aortic rupture in either the thoracic or \nabdominal regions. Surviving males had greatly increased ascending aortic diameters \nand the appearance of pathology at branches of the abdominal aorta. Female mice had \na much lower incidence of death but had greatly increased aortic diameters. Although \nNE infusion also increased systolic blood pressure, it did not significantly augment \nmortality or aortic diameters. MicroCT discerned novel pathology during AngII infusion \nthat included development of branch aneurysms in the celiac and superior mesenteric \narteries. \nConclusion: \nAngII greatly enhanced aortic pathology in Fbn1\nC1041G/+ mice, manifested by \naortic rupture in both the thoracic and abdominal regions and development of \npathologies at aortic branches of the celiac and superior mesenteric arteries. \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n4 \n \nINTRODUCTION \nFibrillin-1 (FBN1) is a large protein (~350 kDa) that is hypothesized to be \nrequired for development of a scaffold on which elastic fibers are developed. Genetic \nvariants of FBN1 are the underlying cause of Marfan syndrome. The manifestations of \nMarfan Syndrome are highly diverse, which may be at least partially attributable to the \nfact that there are over 2,000 variants.1 While attention has commonly focused on the \naberrant dilation of the aortic root in Marfan patients, it is increasingly recognized that \nthese individuals have diverse vascular pathologies. These include aneurysm, \ndissection, and rupture in aortic areas beyond the root, including the ascending, \ndescending thoracic, and abdominal regions.\n2 Aneurysms also frequently occur at \nmultiple aortic branch points. This includes aortic branches arising from the aortic arch \n(brachiocephalic, carotid, subclavian), descending thoracic (vertebral, bronchial), \nsuprarenal (celiac, superior mesenteric, renal), and infrarenal regions (iliac).\n3 The \npresence of ancillary aneurysms further increased the complexity of pathologies \ninduced by FBN1 variants.  \n \nWhile FBN1 variants cause a diversity of vascular phenotypes, experimental \nmodels using FBN1 variants are predominantly focused on the proximal thoracic aorta.\n4 \nThe most commonly used model is mice expressing a Fbn1C1041G/+ variant.5 These mice \nhave a modest expansion of the aortic root and ascending aorta,6, 7 which exhibits \nprofound sexual dimorphism, with progressive enlargement only occurring in male mice \nup to one year of age.\n7, 8 There have been no reports of aortic dissection or rupture in \nFbn1 C1041G/+ mice. \n  \nTo determine the role of angiotensin II (AngII) in arterial diseases, many studies \nhave employed chronic subcutaneous infusions, most commonly at a rate of 1,000 \nng/kg/min.\n9 This procedure in wild type mice promotes perivascular fibrosis in many \narterial beds.10 When combined with hyperlipidemia and BAPN administration, AngII \ninfusion promotes localized pathologies with augmentation of proximal thoracic \naortopathy and progressive expansion and rupture of the suprarenal aorta.\n11-13 AngII \nhas been infused into Fbn1C1041G/+ mice. This includes very high infusion rates of AngII \n(4.5 mg/kg/d = 3,125 ng/kg/min) that increased ascending aorta diameters, with some \nstudies having all mice die of ascending aortic rupture within 4 weeks of infusion.\n14-16 \nAngII infusion at 1,000 ng/kg/min in Fbn1 C1041G/+ mice in normolipidemic background17 \nor deficient in ApoE also resulted in increased aortic aneurysms in both the ascending \nand abdominal regions.\n18 \n \nThe role of increased AngII activation in aortic pathology has not been explored \nextensively in Fbn1C1041G/+ mice. Therefore, this study was designed to address the \nfollowing questions: 1. Whether the sexual dimorphism of vascular diseases that occur \nin Fbn1 \nC1041G/+ mice is retained under AngII activation. 2. Whether pathologies develop \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n5 \n \nbeyond the proximal thoracic aorta in a mode that recapitulates the human disease. 3. \nWhether blood pressure per se augments vascular diseases or whether the effect is \nspecific to AngII activation. \n \nMETHODS \n \nData Availability \nDetailed materials and methods are available in this manuscript. Numerical data \nare available in the Supplemental Excel File. \n \nMice \nStudies were performed in accordance with recommendations for design and \nreporting of animal aortopathy studies.\n19 All experiments used littermate controls. \nFbn1C1041G/+ (stock #012885) mice were obtained from The Jackson Laboratory as \ndescribed previously.8 Male and female Fbn1C1041G/+ mice were bred to C57BL/6J \nfemale and male mice, respectively, to generate male and female Fbn1+/+ and \nFbn1C1041G/+ littermates. Littermates were separated by sex and randomly assigned to \nhousing groups after weaning. Mice were housed up to 5 per cage, maintained on a 14-\nhour light/10-hour dark cycle, fed Teklad Irradiated Global 18% Protein Rodent Diet # \n2918 ad libitum, and allowed ad libitum access to water via a Lixit system. Bedding was \nprovided by P.J. Murphy (Coarse SaniChip) and changed weekly during the study. \nCotton pads were provided as enrichment. The room temperature was maintained at \n21-23 °C, and the humidity was maintained at ~ 50%. \n \nBoth male and female mice were studied. All experiments were approved by the \nUniversity of Kentucky IACUC (Protocol # 2018-2967). \n \nGenotyping \n Mice were genotyped after weaning and termination, respectively, using tail \ntissue: group allocation was based on genotyping performed after weaning at postnatal \nday 28, and the genotype was confirmed again using tissue acquired at the termination \nof each study. Fbn1\nC1041G/+ was assayed using forward primer (5'-\nCTCATCATTTTTGGCCAGTTG-3') and reverse primer (5'-GCACTTGATGCA CATTCA \nCA-3') covering a loxP-flanked neomycin resistance cassette placed in intron 24, which \nis not present in wild type mice. The protocol used was as described by The Jackson \nLaboratory. Fbn1\n+/+ generates a 164 bp product. Fbn1C1041G/+ generates a 212 bp \nproduct. Post-termination validation genotyping was performed by Transnetyx in a \nblinded manner. \n \n \n \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n6 \n \nSubcutaneous Infusions  \n After random assignment, AngII (1,000 ng/kg/min dissolved in saline; Product # \n4006473; CAS # 4474-91-3; Bachem) or its vehicle (saline), or norepinephrine (NE 5.6 \nmg/kg/day; dissolved in saline containing 0.2% wt/vol ascorbic acid20) or its vehicle \n(0.2% wt/vol ascorbic acid in saline) was infused through a subcutaneously implanted \nosmotic pump (ALZET LLC). Alzet model 2001 was used for 3 days and model 1004 for \n28 days of infusion, respectively, in mice at 10 to 14 weeks of age.\n21 Surgical staples \nused to close incision sites were removed 7-10 days after surgery. Postoperative pain \nwas alleviated by application of a topical lidocaine cream (4% wt/wt; Cat # 59-930, \nHealthWise). \n \nSystolic Blood Pressure Measurements \n Systolic blood pressure was measured on conscious mice by a non-invasive tail-\ncuff system (MC4000 Multi Channel system, Hatteras Instruments) following our \nstandard protocol.22 Data were collected at the same time each day for 3 consecutive \ndays. Criteria for accepted data were systolic blood pressure between 70 and 200 \nmmHg and standard deviation < 30 mmHg for at least 5 successfully recorded \ndata/mouse/day. The mean systolic blood pressure of each mouse from the 3-day \nmeasurements was used for data analysis. \n \nNecropsy \n All study mice were checked at least once every day. Necropsies were performed \nimmediately to determine the cause of death after carcasses were found. Aortic rupture \nwas defined as the presence of extravascular blood that accumulated in the body \ncavity. The location of blood egress was determined by the location of the blood clot and \na discernible disruption of the aortic wall. \n \nMicro Computed Tomography (microCT) \nMicroCT was performed as described previously.\n23 Mice were euthanized by an \noverdose of ketamine and xylazine cocktail (90 and 10 mg/kg, respectively). The \nthoracic cavity was cut open, and the right atrium was nicked to allow the exit of blood \nflow. Saline (5 ml) was perfused through the left ventricle. The right atrium was sealed \nusing superglue immediately after perfusion, and Microfil® (Flow Tech, Inc.) was \ninjected through the same catheter. Once Microfil was visualized in the arterioles \nsurrounding the small intestine, the catheter was clamped shut to prevent backflow of \nMicrofil® into the thoracic cavity, and the animal was set aside to allow the compound to \nharden for ~90 minutes.  \n \nAfter Microfil perfusion, animals were scanned using a Skyscan 1276 MicroCT \n(Bruker) or a U-CT Optical Imaging system (MILabs, Netherlands). CT-scanned images \nwere reconstructed using the N-Recon program (Bruker, Belgium) or MILabs \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n7 \n \nreconstruction program (MILabs, Netherlands) to adjust for beam hardening and ring \nartifacts. 3D reconstruction and measurements were performed using the 3D slicer \nprogram. All bones and vasculatures not of interest were removed using a scissor tool \nwithin the program to display the aorta and its major branches. To visualize branch \narteries, all the other surrounding vasculatures were removed using the scissor tool. \n \nMeasurement of in situ Aortic Diameters \nMice were terminated by overdose of ketamine and xylazine mixture followed by \ncardiac puncture and saline perfusion. The order in which mice were terminated was \nrandomized. Aortas were dissected away from the surrounding tissue. A black plastic \nsheet was inserted beneath the aorta and heart to increase contrast and facilitate \nvisualization of aortic borders.\n24 Optimal Cutting Temperature compound (Sakura \nFinetek) was introduced into the left ventricle to maintain aortic patency before imaging. \nAortas were imaged using a Nikon SMZ25 stereoscope, and measurements were \nrecorded using NIS-Elements AR 5.11.03 software (Nikon Instruments Inc.). Ascending \naortic diameters were measured at the largest width perpendicular to the vessel. \n \nPathology \n Thoracic aortas with major branches were harvested from surviving mice after 3 \ndays of AngII infusion and immersed in neutrally buffered formalin (10% wt/vol). Aortas \nwere embedded in OCT and sectioned with a cryostat. \nAbdominal aortas with major branches were dissected free and immersed in \nneutrally buffered formalin (10% wt/vol) overnight, followed by a series of dehydration \nsteps in increasing concentrations of ethanol. The aortas were then embedded in \nparaffin wax. Tissue sections (5 \nμ m) were collected with a microtome. Paraffin-\nembedded sections (5 µm) were deparaffinized using limonene (Cat # 6533A, Medical \nChemical Corporation). \nHematoxylin and eosin (H&E, Cat # 26043-06, Electron Microscopy Sciences; \nCat # AB246824, abcam) and Verhoeff iron hematoxylin staining were performed, \nrespectively, to visualize pathologies and elastic fibers. Picrosirius red/methyl green \nstaining was performed to visualize collagen fibers. Immunostaining was performed \nusing primary and secondary antibodies listed in the Supplemental Materials Major \nResources Tables. NovaRed (Cat #SK-4805, Vector) was used as chromogen. Images \nof histological staining and immunostaining were captured using an Axioscan 7 (Zeiss) \nor Nikon Eclipse Ni and imaged using ZEN v3.1 blue edition (Zeiss) or NIS-Elements \nAR 5.11.03 software (Nikon Instruments Inc) \n \nStatistical Analyses \nSigmaPlot version 15 or 16 (SYSTAT Software Inc.) was used for statistical \nanalysis. Data were presented as either mean ± SEM or median with the 25\nth and 75th \npercentiles, depending on whether the data were analyzed by parametric or non-\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n8 \n \nparametric tests. Normality and homogeneous variance assumptions for data with \nn≥ 6/group were assessed using the Shapiro-Wilk test and the Brown-Forsythe test, \nrespectively. Student’s t-test was used for the data that met both normality and \nhomoscedasticity to compare means. For data that did not pass either the normality or \nequal variance test or n<6/group, Mann-Whitney U-test was applied. Statistical \nsignificance was set at P<0.05. \n \nRESULTS \n \nAngII Infusion Reduced Survival in Male Fbn1\nC1041G/+ Mice \nInfusion of AngII for 3 days led to distinctive blood accumulation in both the \nascending and descending thoracic aorta of Fbn1C1041G/+, but not Fbn1+/+, mice. This \nphenotype was restricted to the outer margins of the media (Figure S1), as has been \ndemonstrated in several other modes of inducing thoracic aortic pathology in mice.21, 25 \n \nAngII infusion into male Fbn1\nC1041G/+ mice for 28 days led to a high incidence of \ndeath (~65%) that was significantly higher compared to the mortality rate in Fbn1+/+ \nmice (Figure 1A). Necropsies of all mice that died within 14 days of AngII infusion \nrevealed characteristic blood accumulation in either the thoracic or abdominal cavity, \nwith approximately equivalent incidence. Since the precise loci of blood egress could \nnot be reliably identified, rupture sites were categorized broadly as thoracic or \nabdominal (Figure 1B). All the deaths due to abdominal aortic rupture occurred within \nthe initial 7 days of AngII infusion. Deaths due to thoracic aortic rupture were also \nprominent within the first 7 days of AngII infusion, but some occurred during more \nprotracted infusion. Some deaths occurred late during AngII infusion that were not \ndiscernible as being due to loss of vascular integrity (Supplemental Table 1). In contrast \nto the high mortality observed in male mice, female Fbn1\nC1041G/+ mice exhibited a low \nincidence (20%) of death during AngII infusion, which did not differ significantly from that \nof Fbn1\n+/+ mice (Figure 1C). All the vascular deaths in females were attributed to \nthoracic aortic rupture (Figure 1D). \n \nAortas were visualized in mice that survived 28 days of AngII infusion, and in situ \ndiameters were measured as described previously.\n24 Aortas of both Fbn1+/+ and \nFbn1C1041G/+ male mice infused with saline had a similar opacity, which was increased \nfollowing AngII infusion (Figure 1E). AngII infusion significantly increased aortic \ndiameters in both Fbn1\n+/+ and Fbn1C1041G/+ male mice (Figure 1F). Female Fbn1+/+ and \nFbn1C1041G/+ mice also had similar opacity during saline infusion (Figure 1G). AngII \ninfusion into Fbn1+/+ female mice failed to promote an overt change in opacity but \ndiameters were increased modestly. AngII infusion into female Fbn1C1041G/+ mice \npromoted grossly discernible pathology in the ascending aorta and pronounced \nincreases in diameters (Figure 1H).  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n9 \n \n \nAortas were dissected free to determine whether pathologies were present in any \nother regions of the aorta. During this tissue processing, longitudinal tortuosity in \nFbn1C1041G/+ mice infused with AngII was noted. Therefore, aortic lengths were \nmeasured from the branch of the subclavian to the iliac bifurcation. AngII infusion \nsignificantly increased the length of the aorta, irrespective of Fbn1 genotype (Figure \n1K). The length of the aorta was much greater in male Fbn1C1041G/+ mice and was \nsignificantly increased during AngII infusion (Figure 1K). In contrast, the aortic length \nwas not different between saline and AngII infusion in female Fbn1C1041G/+ mice. \n \nNorepinephrine Increased Blood Pressure but Failed to Induce Pronounced Aortic \nDisease  \n To determine whether increased systolic blood pressure induced by AngII \ninfusion augmented aortic pathologies in Fbn1C1041G/+ mice, this strain was infused with \nNE. Infusion of NE at a rate of 5.6 mg/kg/day led to significant increases in systolic \nblood pressure in both male and female Fbn1+/+ and Fbn1C1041G/+ mice (Figure S2A and \nB). In contrast to AngII infusion, there were no deaths in either male or female Fbn1+/+ or \nFbn1C1041G/+ mice during NE infusion (Figure S2C and D). Also, there were no apparent \nchanges in the in situ appearance of aortas during NE infusion in either male or female \nFbn1\n+/+ and Fbn1C1041G/+ mice (Figure S2E and G), nor in the maximum external \ndiameter of the ascending aorta (Figure S2F and H). Furthermore, the increased \nsystolic blood pressure induced by NE infusion failed to alter aortic length or develop \nany grossly apparent aortic pathologies (Figure S2I-L). \n \nAngII Promoted Development of Abdominal Aortic Branch Aneurysms in \nFbn1\nC1041G/+ Mice \nEx vivo examination of aortas revealed the development of aortic branch \naneurysms in the abdominal region of AngII-infused Fbn1C1041G/+ mice. To gain further \ninsight, aortic imaging was performed using microCT in groups of saline and AngII-\ninfused mice, respectively. No abdominal aortic branch aneurysms were detected in \nmale and female Fbn1\n+/+ and Fbn1C1041G/+ mice following infusion with saline, or in \nFbn1+/+ male and female mice infused with AngII (Figure 2A and S3A). In contrast, AngII \ninfusion into Fbn1C1041G/+ male and female mice produced marked aneurysmal \nexpansion in the celiac and superior mesenteric arteries, while not affecting the left or \nright renal arteries (Figure 2B and S3B). The increased aneurysm at the aortic branches \nof the celiac and superior mesenteric arteries was a highly consistent pathology in \nFbn1\nC1041G/+ mice (Figure 2, S3, and Videos 1 and 2). The pathology of aneurysm at \nboth the celiac and superior mesenteric arterial branches was characterized by \ncomparable degrees of elastic fragmentation and regeneration, collagen deposition, \nexpansion of the number of cells expressing α -smooth muscle cell actin, and the \npresence of numerous CD68 positive cells (Figures 2C and S4), when compared to \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n10 \n \ntissues from the same regions of Fbn1+/+ and Fbn1C1041G/+ mice infused with saline or \nFbn1+/+ mice infused with AngII. \n \nDISCUSSION \nThere is an increasing awareness that FBN1 variants can lead to a spectrum of \naortic pathologies. For example, it has been recognized recently that branch aneurysms \nare frequent in Marfan Syndrome patients and are associated with vascular risk.3 This \nstudy has demonstrated that infusion of AngII into mice expressing the C1041G variant \nof Fbn1 leads to augmented aneurysms in the proximal thoracic aorta, accelerated \naortic dissection and rupture, and aortic branch aneurysms. \n \nOur previous studies demonstrated the age-dependent expansion of the \nascending aorta in the Fbn1C1041G/+ has a strong sexual dimorphism, with males \nshowing gradual dilation over the course of a year, whereas females display a rate of \nincrease indistinguishable from that of Fbn1\n+/+ mice.8 These results were replicated in \nthe current study. Sexual dimorphism was also present in some aspects of aortic \ndiseases during AngII infusion. Specifically, there was a much greater incidence of \ndeath due to loss of aortic integrity in males compared to females. However, similar to \nmales, female Fbn1\nC1041G/+ mice infused with AngII had striking expansion of the \nascending aorta. This finding implies that distinct mechanisms underlie aneurysm \nformation and rupture in Fbn1C1041G/+ mice, which will need to be further elucidated. \n \nThe basis for AngII promoting aortic diseases in a regional manner is unclear. \nThere have been several proposed mechanisms for the regional specificity of aortic \ndisease, with a major emphasis on embryonic origins of smooth muscles cells.\n26, 27 It \nhas been established that AngII-induced aortic pathologies are due to stimulation of \nAT1a receptors.\n28 However, deletion of AT1a receptors in aortic smooth muscle cells \nhas not been demonstrated to influence aortic diseases, while its deletion in \nendothelium has a modest reduction compared to whole body deletions.29-31 Given that \nthe aortic adventitia is heterogeneous in its composition throughout the length of the \nvessel, this is a potential basis for this regional heterogeneity. Region-specific cell types \ninclude perivascular adipocytes and fibroblasts.\n32, 33 Resolving the contributions of \nadventitial components to aortic pathology will require the development of mice \nexpressing Cre under the control of promoters that specifically target these cell types. \n \n A novel observation in this study is the presence of aortic branch aneurysms \npredominantly involving the celiac and superior mesenteric arteries in AngII-infused \nFbn1\nC1041G/+ mice, irrespective of sex. This finding parallels recent clinical reports \ndemonstrating aortic branch aneurysms in patients with Marfan syndrome.3 Consistent \nwith these observations, the 2022 ACC/AHA guidelines recognize that aortic branch \naneurysms are more common than previously appreciated, with an elevated likelihood \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n11 \n \nof requiring aortic surgery.34 The mechanisms by which AngII preferentially promotes \naneurysm formation in the celiac and superior mesenteric arteries of Fbn1C1041G/+ mice \nremain unclear. Both arteries supply blood to major gastrointestinal organs, and the \nsuperior mesenteric artery functions as a resistant artery critical for regulating regional \nblood flow and systemic blood pressure. Their unique hemodynamic demands, complex \nbranching geometry, and distinct extracellular matrix architecture may render these two \narteries susceptible to the combined effects of Fbn1 haploinsufficiency and AngII-\ninduced mechanical and inflammatory stress. Given their central roles in abdominal \norgan perfusion, further exploration is warranted to elucidate how AngII drives \naneurysmal development and vascular remodeling in these specific arteries of \nFbn1\nC1041G/+ mice. \n \nInfusion of AngII at sufficient rates increases systolic blood pressure, which may \ncontribute to aortic pathology. One strategy to determine the role of blood pressure has \nbeen to compare pathology in mice infused with either AngII or NE at rates that produce \nequivalent increases in systolic blood pressure. Using this approach, blood pressure per \nse has not been implicated in the development of atherosclerosis,\n35 abdominal aortic \naneurysm,36 or ascending aortic aneurysms.20 In this study, NE increased systolic blood \npressure but failed to replicate the aortic pathologies generated during AngII infusion \nwhich implies that increased blood pressure is not a sufficient cause of the disease. \nAngII increases systolic blood pressure, which may contribute to the aortic pathology. \n \nIn summary, this study demonstrates that AngII infusion in Fbn1\nC1041G/+ mice \naugments aneurysmal disease in the distal thoracic aorta and induces previously \nundocumented pathologies, including aortic rupture and aortic branch aneurysms. This \nmodel provides a valuable platform to perform subsequent studies to define the \nmechanisms of these diverse pathologies. \n \nAcknowledgments \n These studies were facilitated by the University of Kentucky Light Microscopy \nCore (RRID:SCR 026405) and the Magnetic Resonance and Spectroscopy Core \n(RRID:SCR 026383). \n  \nSources of funding \n This research work is supported by the National Heart, Lung, and Blood Institute \nof the National Institutes of Health (R35HL155649, K01HL149984), a Merit award from \nthe American Heart Association (23MERIT1036341), and a Leducq Foundation Network \nof Excellence (22CVD03).  \n \nDisclosures \n The authors have no conflicts of interest.   \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n12 \n \nFIGURE LEGENDS \nFigure 1.  AngII Infusion Augmented Ascending Aortic Aneurysm in Fbn1C1041G/+ \nmice and Induced Aortic Rupture. Ten-14-week-old male and female \nFbn1+/+ and Fbn1C1041G/+ mice were infused with either saline or AngII for \n28 days. Survival curves and cause of death in male (A-B) and female (C-\nD) mice. P values (A, C) were determined by Log-Rank analysis. \nRepresentative in situ images of the thoracic aorta and maximal diameters \nof ascending aortas in male (E-F) and female (G-H) mice. Representative \nex vivo images of the entire aorta in male (I) and female (J) mice. Aortic \nlength measured from the left subclavian branch to the iliac bifurcation in \nmale (K) and female (L) mice. Data in (F, H, L) were analyzed using \nMann-Whitney Rank Sum test, and data in (F, K) were analyzed using \nStudent’s t-test. \n \nFigure 2. AngII Promoted Development of Aortic Branch Aneurysms in \nFbn1\nC1041G/+ Mice. Ten-14-week-old male Fbn1+/+ and Fbn1C1041G/+ mice \nwere infused with either saline or AngII for 28 days. (A) Representative \nmicroCT images of the entire aorta and the abdominal aorta. (B) Maximum \ndiameters of the aortic branch points for celiac, superior mesenteric, right \nrenal, and left renal arteries measured using microCT images. Statistical \nanalysis was performed using Mann-Whitney Rank Sum test. (C) Tissue \nsections of the celiac and superior mesenteric aortic branches were \nstained with Verhoeff’s iron hematoxylin, picrosirius red/methyl green, and \nimmunostained for α -smooth muscle actin and CD68, respectively. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n13 \n \nSupplemental Figure Legends \n \nFigure S1. AngII Infusion Led to Rapid Formation of Thoracic Aortic Dissection. \nIn situ images of the ascending and descending thoracic aorta of male \nFbn1\n+/+ and Fbn1C1041G/+ mice infused with AngII for 3 days. \n \nFigure S2. NE Did not Promote Evident Aortic Pathologies in Fbn1\n+/+ and \nFbn1C1041G/+ Mice. Ten-14-week-old male and female Fbn1+/+ and \nFbn1C1041G/+ mice were infused with either vehicle (0.2% wt/vol ascorbic \nacid dissolved in saline) or NE for 28 days. Systolic blood pressure \nmeasurements in male (A) and female (B) mice. Survival curves for male \n(C) and female (D) mice. Representative in situ images of the thoracic \naorta and maximal diameters of ascending aortas in male (E-F) and \nfemale (G-H) mice. Representative ex vivo images of the entire aorta in \nmale (I) and female (K) mice. Aortic length measured from the left \nsubclavian branch to the iliac bifurcation in male (J) and female (L) mice. \nData in (A, F, H, J, and Fbn1+/+ mice in B and L) were analyzed using \nMann-Whitney Rank Sum test, and data in Fbn1C1041G/+ mice (B, L) were \nanalyzed using Student’s t-test. \n \nFigure S3. AngII Infusion Promoted Development of Abdominal Aortic Brach \nAneurysms in Female Fbn1C1041G/+ Mice. Ten-14-week-old female \nFbn1+/+ and Fbn1C1041G/+ mice were infused with either saline or AngII for \n28 days. (A) Representative microCT images of the entire aorta and the \nabdominal aorta. (B) Maximum diameters of the aortic branch points for \nceliac, superior mesenteric, right renal, and left renal arteries measured \nusing microCT images. Statistical analysis was performed using Mann-\nWhitney Rank Sum test. \n \nFigure S4. Pathological Characterization of the Celiac and Superior Mesenteric \nArteries. Ten-14-week-old female Fbn1\n+/+ and Fbn1C1041G/+ mice were \ninfused with either saline or AngII for 28 days. Arterial tissue sections were \nstained with Verhoeff’s iron hematoxylin, picrosirius red/methyl green, and \nimmunostained for α -smooth muscle actin and CD68, respectively. \n \nVideo 1. MicroCT Images of Saline-infused Fbn1+/+ and Fbn1C1041G/+ Male Mice.  \n \nVideo 2. MicroCT Images of AngII-infused Fbn1 +/+ and Fbn1C1041G/+ Male Mice. \n  \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\n14 \n \nReferences \n1. Milewicz DM, Braverman AC, De Backer J, Morris SA, Boileau C, Maumenee IH, \nJondeau G, Evangelista A and Pyeritz RE. Marfan syndrome. Nat Rev Dis Primers. \n2021;7:64. \n2. Requejo-Garcia L, Martinez-Lopez R, Plana-Andani E, Medina-Badenes P , \nHernandiz-Martinez A, Torres-Blanco A and Miralles-Hernandez M. Extrathoracic \naneurysms in Marfan syndrome: A systematic review of the literature. Ann Vasc \nSurg. 2022;87:548-559. \n3. 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It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\nFigure 1\nMale\nCause of Death\nThor Abd Non-\nVasc\n20\n40\n60\n80\n100\nMale\nin situ\nMale\nSurvival\n10 20 280 Days\n20\n40\n60\n80\n100% SurvivalFbn1+/+ Saline\nFbn1+/+ AngII\nFbn1C1041G/+ Saline\nFbn1C1041G/+ AngII\nP<0.001\nFbn1+/+ Fbn1C1041G/+\nMale\nThoracic Aorta\nMaximal Diameter (mm)0.5\n1.0\n1.5\n2.0\n2.5\n3.0\n3.5\nSaline AngII Saline AngII\nFbn1+/+\n10 20 280\nDays\n% of Total\nFemale\nin situ\nFemale\nThoracic Aorta\nFbn1+/+ Fbn1C1041G/+\n3.0\n4.0\n3.5\n2.0\n0.5\n1.5\n2.5\n1.0\nFbn1+/+\nSalineSaline AngII AngII\n20\n40\n60\n80\n100\nMale\nex vivo\nMale\nex vivo\nFemale\nex vivo\nFemale\nex vivo\nFbn1+/+\nAngII AngIISaline Saline\nFbn1+/+\nSalineSaline AngIIAngII\n40\n35\n30\n25\nSalineSaline AngII AngII\nFbn1+/+\nFbn1+/+ Saline\nFbn1+/+ AngII\nFbn1C1041G/+ AngII\nFbn1C1041G/+ Saline\nP=0.0036P<0.001\nP=0.0050\nBA C\nE GF H\nI KJ L\nSalineSalineAngII\nFbn1+/+\n40\n35\n30\n25Aortic Length (mm)\nP<0.001 P=0.0068\nAngII\nP=0.012\nP=0.009 P>0.99\nP=0.15\nD\nFbn1C1041G/+ Fbn1C1041G/+\nFbn1C1041G/+Fbn1C1041G/+\nFbn1C1041G/+Fbn1C1041G/+\n% Survival\nThor Non-\nVasc\n20\n40\n60\n80\n100\nSaline\nAngII\nSaline\nAngII\n% of TotalMaximal Diameter (mm)\nAbd\nFemale\nCause of Death\nFemale\nSurvival\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint \n\nFbn1C1041G/+Fbn1 +/+\nAngII\nSaline\nFigure 2\nRight Renal Left Renal\nFbn1+/+Fbn1+/+ Fbn1C1041G/+ Fbn1C1041G/+\nA\nB\nC\nSMA\nCeliac\nPicro.Red/Meth.Green α-SMA CD68\n200 µm\nSalineSaline AngII AngII SalineSaline AngII AngII\nFbn1+/+ Fbn1C1041G/+\nP=0.032P>0.99\nSalineSaline AngII AngII\nCeliac\n2.0\n1.5\n1.0\n0.5\n0.0\nFbn1+/+ Fbn1C1041G /+\nP=0.016P=0.73\nSalineSaline AngII AngII\nP=0.41 P=0.11 P=0.11 P=0.063\nMaximal Diameter (mm)\nVerhoeff’s\nSuperior Mesenteric\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted November 30, 2025. ; https://doi.org/10.1101/2025.11.26.690689doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}