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Background: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (β-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. Methods: BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. Results: Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. Conclusions: Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency ( Fbn1 C1041G/+ ). Approach and Results: Thoracic aortic aneurysm in Fbn1 C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1 C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1 C1041G/+ mice that were either +/+ or −/− for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1 C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1 C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1 C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1 C1041G/+ mice.

BackgroundAngiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and β-sheet regions among species; however, their functions have not been studied.MethodsAdeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), β-sheet (AAV.βsheet-Mut), or both regions (AAV.loop/βsheet-Mut) were injected into male hepatocyte-specific AGT deficient (hepAGT-/-) mice in an LDL receptor –/– background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a Western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells.ResultsIn hepAGT-/– mice infected with AAV.loop-Mut or βsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Surprisingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/– mice infected with AAV.loop/βsheet-Mut were not different from mice infected with AAV.null. In contrast, hepaticAgtmRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated trol and AAV containing wild-type mouse AGT as a positive control. We have demonstra ted consistently in this and previous studies tht located in the endoplasmic reticulum.ConclusionsThe conserved sequences in either the loop or β-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.HIGHLIGHTSThe loop and β-sheet regions in the distal face of angiotensinogen (AGT) have highly conserved sequences across species.Mutations on either the loop or β-sheet regions do not affect plasma AGT concentrations, blood pressure, and atherosclerosis in hypercholesterolemic mice.The conserved sequences in the loop and β-sheet regions regulate the secretion of AGT from hepatocytes synergistically in vivo and in cultured cells.

Vascular smooth muscle cells (vSMC) exert a critical role in sensing and maintaining vascular integrity. These cells abundantly express the low-density lipoprotein receptor-related protein 1 (LRP1), a large endocytic and signaling receptor that recognizes numerous ligands including ApoE-rich lipoproteins, proteases, and protease-inhibitor complexes. We observed the spontaneous formation of aneurysms in the superior mesenteric artery (SMA) of both male and female mice in which LRP1 was genetically deleted in v SMC (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II (AngII)-mediated signaling, suggesting that this pathway is dysregulated. Administration of losartan, an AngII type I receptor antagonist, or an angiotensinogen antisense oligonucleotide to reduce plasma angiotensinogen concentrations restored the normal SMA phenotype in smLRP1-/- mice and prevented aneurysm formation. Additionally, employing a vascular injury model, we noted excessive vascular remodeling and neointima formation in smLRP1-/- mice that was restored by losartan administration. Together, these findings reveal that LRP1 regulates vascular integrity and remodeling of the SMA by attenuating excessive AngII-mediated signaling.

Atherosclerosis is a leading cause of morbidity and mortality in many countries. Mice are the most frequently used animal model to study the pathogenesis and molecular mechanisms of atherosclerosis. En face analyses of the aorta and cross-sections of the aortic root are the two common modes for quantifying the severity of atherosclerosis in mice. This mini-review introduces these two methods, discusses their pros and cons, and provides suggestions to optimize the quantification of atherosclerosis, thereby enhancing rigor and reproducibility in preclinical research.

OBJECTIVE: Liver-derived angiotensinogen (AGT) is the substrate from which renin and ACE generate angiotensin (Ang) II. Ang II not only increases blood pressure, but also is a major determinant of atherosclerosis in hypercholesteremic mice. Here we investigated the effect of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASOs) on blood pressure and atherosclerosis in hypercholesterolemic mice. DESIGN AND METHOD: Eight-week-old male LDL receptor deficient mice were fed a Western diet for 12 weeks, after receiving vehicle, GalNAc control ASO, or 1, 2.5 or 5 mg/kg of GalNAc AGT ASO on day 1, 3, 5 and 7 in the week prior the start of the diet (n = 6-10/group). Treatment was continued thereafter on a weekly basis. Systolic blood pressure (SBP) was monitored by tail cuff. After 12 weeks, mice were euthanized and plasma was collected. Plasma AGT concentration was measured by ELISA kit, and aortic atherosclerotic lesion area was measured by an en face method. RESULTS: The 3 GalNAc AGT ASO doses decreased plasma AGT from 10.3 ± 0.8 to 1.4 ± 0.1, 0.9 ± 0.1 and 0.7 ± 0.1 g/mL, respectively. These decreases were paralleled by dose-dependent SBP reductions from 114 ± 3 to 96 ± 2, 92 ± 2, and 84 ± 2 mmHg, respectively, and atherosclerotic lesion area reductions from 27.4 ± 0.9 to 15.1 ± 1.4, 10.8 ± 1.2, and 7.7 ± 1.0%, respectively. Yet, the attenuation of liver steatosis (liver triglyceride/weight from 139.7 ± 35.7 to 26.8 ± 4.5, 19.9 ± 1.2 and 36.0 ± 3.6 g/mg, liver total cholesterol/weight from 19.8 ± 1.6 to 8.6 ± 0.9, 6.1 ± 0.6 and 8.2 ± 0.7 g/mg) was identical for each GalNAc AGT ASO dose. GalNAc control ASO did not have effects on plasma AGT, SBP, atherosclerosis lesion area, or liver steatosis vs. vehicle. CONCLUSIONS: In hypercholesterolemic mice, inhibition of hepatic AGT reduces systolic blood pressure and atherosclerosis dose-dependently, while liver steatosis was prevented in dose-independent manner.

Objective: Cross-linking of elastin and collagen fibers is a vital process in aortic development. Pharmacological inhibition of aortic cross-linking by β-aminopropionitrile (BAPN) leads to thoracic aortopathies, such as thoracic aortic aneurysm and dissection, in mice. Although the renin angiotensin system (RAS) contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the RAS protects against aortopathy formation caused by the impairment of elastin and collagen cross-linking in mice. Approach and Results: Either vehicle or losartan (15 mg/kg/d, AT1 receptor blocker) were administered through osmotic pumps for 4 weeks in male C57BL/6J mice (3-4-weeks-old, n=30/group). BAPN (0.5% wt/vol) was given through drinking water to induce aortopathies. Losartan increased plasma renin concentrations significantly compared to vehicle-infused mice, indicating effective blockade of at1 receptors. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor, we also tested the effects of irbesartan, an insurmountable inhibitor of AT1 receptor blocker, on BAPN-induced aortopathy formation (50 mg/kg/day, diet, n=30/group). Despite the significant increase of plasma renin concentrations, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin receptor blockade. We next inhibited angiotensinogen, the unique substrate for angiotensin II, using an antisense oligonucleotide targeting angiotensinogen (AGT-ASO). AGT-ASO decreased plasma angiotensinogen concentrations, while aortic death and dilatations were not attenuated. Since hepatocytes are the major source of angiotensinogen, BAPN-induced thoracic aortopathy formation was also examined in mice with hepatocyte-specific angiotensinogen deletion. Although plasma angiotensinogen concentrations displayed 90% reduction compared to wild type littermates, aortic rupture and aneurysm formation were not suppressed. Conclusion: Inhibition of the RAS does not attenuate BAPN-induced thoracic aortopathy formation in mice.

Objective: Angiotensin-converting enzyme (ACE) generates Angiotensin II (AngII), the bioactive angiotensin peptide that contributes to atherosclerosis. ACE is present in proximal tubular cells (PTCs), and AngII concentrations are higher in kidney than in plasma. The purpose of this study was to determine whether ACE in PTCs contributed to atherosclerosis in both male and female mice. Approach and Results: ACE f/f x Ndrg1-Cre ERT2 -/- (PTC-ACE+/+) and ACE f/f x Ndrg1-Cre ERT2+/- (PTC-ACE -/-) mice were generated by breeding female ACE floxed mice (ACE f/f) xNdrg1-Cre ERT2-/- and male ACE f/f x Ndrg1-Cre ERT2+/- transgenic mice. Both male and female littermates in an LDL receptor -/- background were used to study atherosclerosis. PTC-ACE+/+ and PTC-ACE-/- littermates were administered with of tamoxifen (150 mg/kg/day) for 5 consecutive days at the age of 4-6 weeks to ACE deletion in PTCs. Two weeks after the last injection of tamoxifen, mice were fed a Western diet for 12 weeks. Blood pressure was measured using a tail-cuff system during the study. Systolic blood pressure was not different between the two genotypes (PTC-ACE+/+ vs -/-: 124±3 vs 121±2 mmHg in male, P=0.3; and 114±3 vs 115±3 mm Hg in female, P=0.9). All mice were hypercholesterolemic, and plasma cholesterol concentrations were not different between the two genotypes (PTC-ACE +/+ vs -/-: 1524±108 vs 1680±66 mg/dl in male, P=0.2; 1175±47 vs 1210±55 mg/dl in female, P=0.6). ACE mRNA abundance measured by qPCR of ACE in kidneys was less in the PTC-ACE-/- mice (PTC-ACE +/+ vs -/-: 1.03±0.11 vs 0.33±0.04 in male, P=0.001; 1.14±0.21 vs 0.64±0.09 in female, P=0.02). In PTC-ACE-/- mice, immunostaining of ACE in kidney sections showed that ACE was deleted in S1 and S2 PTCs, but remained in S3 of the outer medulla. Atherosclerosis was measured using an en face method. PTC-ACE+/+ and PTC-ACE-/- mice had comparable atherosclerotic lesion size in the ascending and aortic arch regions (PTC-ACE+/+ vs -/-: 11.4±1.4% vs 13.4±1.7% in male, P=0.4; 14.9±2.2% vs 15.7±3.7% in female, P=1.0). Conclusion: PTC-specific ACE deficiency did not affect atherosclerosis in hypercholesterolemic mice.

Objective: This study determined whether angiotensinogen (AGT) interacted with renin in renal proximal tubule cells (PTCs) to promote atherosclerosis. Approach and Results: Since hepatocyte-produced AGT can be filtered by glomeruli and retained in renal PTCs, we first determined whether hepatocyte-derived AGT interacts with renin in PTCs to promote atherosclerosis. Transgenic mice expressing human renin in PTCs driven by a kidney androgen-related protein promoter (KAP-hREN) in an LDLR -/- background were used. To induce synthesis of human AGT in hepatocytes, an adeno-associated viral vector (AAV) containing human AGT with a liver-specific promoter was injected intraperitoneally. Three groups of male littermates were administered testosterone to activate human renin expression in PTCs: (1) wild type mice administered null AAV, (2) KAP-hREN transgenic mice administered null AAV, and (3) KAP-hREN transgenic mice administered AAV containing human AGT. Two weeks after administration of testosterone and AAVs, mice were fed a Western diet for 6 weeks. Induction of human AGT in liver and human renin in PTCs did not increase atherosclerosis, comparing to the other two groups. Next, to evaluate the direct interaction of AGT and renin in PTCs for promoting atherosclerosis, KAP-human AGT (KAP-hAGT) and KAP-hREN double transgenic mice were fed a Western diet for 12 weeks. Although immunostaining confirmed the presence of human AGT and human renin in PTCs, double transgenic mice did not have increased percent atherosclerotic lesion area, comparing to either wild type or single transgenic littermate groups. Surprisingly, retired male double transgenic breeders (46-67 weeks old) showed severe atherosclerosis spontaneously without Western diet feeding, while their single transgenic littermates had minimal to modest atherosclerotic lesions. The sample size was small so statistical analysis was not performed. Conclusions: The presence of human AGT in liver or PTCs with combination of human renin in PTCs did not augment Western diet-induced atherosclerosis in mice. Further studies with increased numbers are needed to determine whether retired male breeders containing human AGT and human renin in PTCs develop severe atherosclerosis.

Objective: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT −/− ) mice in an LDL receptor–deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT −/− mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT −/− mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT −/− mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT +/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT −/− mice to the magnitude of hepAGT +/+ mice. Conclusions: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor–deficient mice.

BACKGROUND: Angiotensin (Ang)I is cleaved by angiotensin-converting enzyme (ACE) to generate AngII. The purpose of this study was to determine the roles of ACE in endothelial and smooth muscle cells in aortic aneurysms. METHODS AND RESULTS: AngI infusion led to thoracic and abdominal aortic aneurysms in low-density lipoprotein receptor-deficient mice, which were ablated by ACE inhibition. Endothelial or smooth muscle cell-specific ACE deletion resulted in reduction of AngI-induced thoracic, but not abdominal, aortic dilatation. CONCLUSIONS: AngI infusion causes thoracic and abdominal aortic aneurysms in mice. ACE in aortic resident cells has differential effects on AngI-induced thoracic and abdominal aortic aneurysms.

Background: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies. Methods: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)–infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry–assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor–related protein 1) or Tgfbr2 (transforming growth factor–β receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity. Results: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies. Conclusions: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor–β signaling associated with increases of aortic PAI1.

Hepatocyte-derived angiotensinogen (AGT) is the precursor of angiotensin II (AngII). We determined the effects of hepatocyte-specific (N-acetylgalactosamine-conjugated) antisense oligonucleotides targeting AGT (GalNAc AGT ASO) on AngII-mediated blood pressure (BP) regulation and atherosclerosis and compared its effects with losartan, an AngII type 1 (AT1) receptor blocker, in hypercholesterolemic mice. Eight-week-old male low-density lipoprotein (LDL) receptor deficient mice were administered vehicle or GalNAc AGT ASO (1, 2.5, or 5 mg/kg) subcutaneously beginning 2 weeks before the initiation of Western diet feeding. All mice were fed Western diet for 12 weeks. Their systolic BP was monitored by the tail-cuff technique, and the atherosclerotic lesion area was measured by an en face method. Although the effects of all 3 doses of GalNAc AGT ASO on plasma AGT concentrations were similar, GalNAc AGT ASO reduced BP and atherosclerotic lesion size in a dose-dependent manner. Subsequently, we compared the effects of GalNAc AGT ASO (5 mg/kg) with losartan (15 mg/kg/day). Compared to losartan, GalNAc AGT ASO led to more profound increases in plasma renin and reduction in BP but had similar effects on atherosclerosis. Remarkably, GalNAc AGT ASO also reduced liver steatosis, which was not observed in losartan-treated mice. In conclusion, the BP increase and atherosclerosis development in hypercholesterolemic mice are dependent on AngII generated from hepatic AGT. Deleting hepatic AGT improves diet-induced liver steatosis, and this occurs in an AT1 receptor-independent manner.

Objective: Renin cleaving angiotensinogen to release angiotensin I is the rate-limiting step to produce angiotensin II. Although juxtaglomerular cells are the predominant source of renin in circulation, renin protein is also present in proximal tubules. In this study, we investigated the effects of renin in proximal tubule cells (PTC) on development of atherosclerosis in hypercholesterolemic mice Approach and Result: Inducible PTC-specific renin deficient (PTC-renin -/-) mice were developed by breeding renin floxed mice with mice expressing Cre under the control of the N-myc downstream regulated gene 1 (Ndrg1-Cre ERT2). All study mice were in a low-density lipoprotein receptor (LDLR) deficient background, and both male and female littermates were studied. Littermates of renin floxed mice with or without Ndrg1-Cre ERT2 transgene were injected intraperitoneally with tamoxifen (150 mg/kg/day) for 5 consecutive days when they were 4-6 weeks old. Cre genotypes were determined after weaning (~ 3-4 weeks old) and deletion of renin gene in kidney was verified by PCR of DNA extracted from kidney after termination (~18-20 weeks old). Two weeks after the last injection of tamoxifen, mice were fed a Western diet for 12 weeks, which led to plasma total cholesterol concentrations above 1000 mg/dl (P = 0.893 in male and P = 0.192 in female between PTC-renin +/+ and -/- mice). PTC-specific renin deficiency did not affect blood pressure (PTC-renin+/+ vs -/-: 110 ± 1 mmHg vs 112 ± 1 mmHg in male, P = 0.452; and 105 ± 3mmHg vs 106 ± 2 mmHg in female, P = 0.875), as measured by a tail-cuff system. Atherosclerosis in the ascending and aortic arch regions was measured with an en face method. No differences on percent lesion area between the two genotypes in both male and female mice (PTC-renin+/+ vs -/-: 18.1 ± 1.4% vs 18.9 ± 1.5% in male, P = 0.315; and 16.2 ± 1.5% vs 16.2 ± 1.2% in female, P = 0.648). Conclusion: Renal proximal tubule-specific renin deficiency does not affect blood pressure and atherosclerosis in hypercholesterolemic mice.

Introduction: Fibrillin-1 ( Fbn1 ) mutations are associated with thoracic aortic aneurysms (TAAs) in patients with Marfan syndrome. Constitutive deletion of Fbn1 leads to neonatal death due to aortic rupture in mice. However, the role of Fbn1 in maintaining aortic integrity during the adult phase remains unclear. Since smooth muscle cell (SMC) is a major constituent of the aortic media, this study examined whether deletion of Fbn1 in SMCs of adult mice leads to compromise of aortic integrity. Methods and Results: Fbn1 floxed mice were generated that included insertion of tdTomato as a reporter for gene deletion. Female Fbn1 floxed mice were bred with male Fbn1 floxed mice expressing CreER T2 driven by an Acta2 promotor. Tamoxifen was injected intraperitoneally for 5 days into male Fbn1 floxed littermate mice that were either Cre negative or positive (n=6-8/genotype, 5-week-old) to create sm Fbn1 +/+ and -/- mice, respectively. Gene recombination was confirmed by the presence of tdTomato in sm Fbn1 -/- mice using PCR and confocal microscopy. To investigate the impact of SMC-specific Fbn1 deletion, male sm Fbn1 +/+ and -/- littermates terminated at two intervals, 5 and 36 weeks after completion of tamoxifen injections. In situ aortic images were captured, and aortic diameters were measured at ascending, arch, and descending thoracic aortas. Five weeks after tamoxifen injection, aortic diameters of smFbn1+/+ and -/- mice were not different. Despite the long-term deletion of Fbn1 in SMCs, aortic dilatation was not observed in any regions of aortas in sm Fbn1 -/- mice compared to wild type littermates 36 weeks after tamoxifen injection. Next, we investigated whether Fbn1 deletion in SMCs augmented angiotensin II (AngII)-induced aortic dilatation. AngII was infused subcutaneously into male sm Fbn1 +/+ and -/- littermates for 12 weeks (n=11-12/genotype), started 2weeks after completion of tamoxifen injections. AngII-induced aortic dilatations were augmented modestly by SMC-specific Fbn1 deletion (+/+ 1.7±0.0, -/- 1.9±0.1 mm, P=0.047). Conclusion: Deletion of Fbn1 in SMCs did not cause spontaneous TAA formation and had modest effects on AngII-induced TAAs in adult mice. Fbn1 in SMCs does not exert a critical role in mice after the aortic structure has formed.

Objective: Hepatocyte-produced angiotensinogen (AGT) can be filtered by glomeruli and retained in renal proximal tubules. Renin protein has also been detected in proximal tubules (PTC) of kidney. However, it is unknown whether AGT and renin in proximal tubules contribute to atherosclerosis. This study aimed to determine whether hepatocyte-derived AGT interacts with renin in proximal tubules to promote atherosclerosis. Approach and Results: Transgenic mice expressing human renin driven by a kidney androgen-related protein promoter (KAP-hREN) in an LDLR -/- background were administered subcutaneously with testosterone (15 mg/mouse, constant release for 60 days) to activate the human renin transgene in PTCs. To induce synthesis of human AGT in hepatocytes, adeno-associated viral (AAV; 3 x 10 10 genomic copies/mouse) vector containing human AGT with a liver-specific promoter was injected intraperitoneally. Three groups of male littermates were administered testosterone: (1) wild type mice administered null AAV (n=11), (2) KAP-hREN transgenic mice administered null AAV (n=7), and (3) KAP-hREN transgenic mice (n=8) administered AAV containing human AGT. Two weeks after administration of testosterone and AAVs (either null or human AGT), all mice were fed a Western diet for 6 weeks. Induction of human renin was confirmed by mRNA abundance of human renin in kidney of KAP-hREN transgenic mice. In mice administered human AGT AAV, presence of human AGT was detected in plasma with a human AGT ELISA kit. Induction of both human AGT in liver and human renin in proximal tubules did not affect plasma cholesterol concentrations or systolic blood pressure. Atherosclerotic lesion sizes, as quantified by percent lesion area using an en face method, were not different among the 3 groups (Group 1 vs 2 vs 3: 3.7 ± 0.4% vs 3.3 ± 0.9% vs 2.3 ± 0.3%; P = 0.19). Conclusions: Induction of human AGT in hepatocytes and human renin in proximal tubules does not augment atherosclerosis in hypercholesterolemic mice.

Objective: Angiotensin-converting enzyme (ACE) is the enzyme to generate Angiotensin II (AngII), an important contributor to atherosclerosis. ACE is present in all 3 segments (S1, S2, and S3) of proximal tubular cells (PTCs), and AngII concentrations are higher in kidney than in plasma. The purpose of this study was to determine whether ACE in PTCs contributes to atherosclerosis in mice. Approach and Results: Female ACE floxed mice and male Ndrg1-Cre ERT2 +/- transgenic mice were bred to generate ACE f/f x Ndrg1-Cre ERT2 -/- (PTC-ACE +/+ ) and ACE f/f x Ndrg1-Cre ERT2 +/- (PTC-ACE -/- ) littermates. Male PTC-ACE +/+ (n=7) and PTC-ACE -/- (n=10) littermates in an LDL receptor -/- background were used for atherosclerosis study. After Cre genotyping using tail DNA, mice (both Cre ERT2 -/- and Cre ERT2 +/- ) were injected with 150 mg/kg/day of tamoxifen for 5 consecutive days at the age of 4-6 weeks. Two weeks after the last injection of tamoxifen, mice were fed a Western diet (Envigo, Diet #TD.88137) for 12 weeks to induce hypercholesterolemia. Blood pressure was measured using a tail-cuff system. No difference of blood pressure was detected between the two genotypes (PTC-ACE +/+ vs. PTC-ACE -/- : 126 ± 4 vs. 125 ± 3 mmHg; P = 0.763). All study mice were hypercholesterolemic, but plasma cholesterol concentrations were not different between the two genotypes (PTC-ACE +/+ vs. PTC-ACE -/- : 1376 ± 119 vs. 1543 ± 93 mg/dl; P = 0.28). PTC-specific deletion of ACE was confirmed after termination using immunostaining of ACE. In mice with Ndrg1-Cre ERT2 transgene, immunostaining of ACE in kidney section showed an absence of ACE in S1 and S2 of PTCs, but ACE protein in S3 remained. Deletion of ACE in S1 and S2 of PTCs did not change hypercholesterolemia-induced atherosclerosis in the ascending and aortic arch regions (PTC-ACE +/+ vs. PTC-ACE -/- : 9.6 ± 0.9% vs. 7.0 ± 1.9%; P = 0.13 by Mann-Whitney Rank Sum Test), as measured using an en face method. Conclusion: ACE deficiency in S1 and S2 of renal proximal tubular cells had no effect on atherosclerosis in hypercholesterolemic mice. Since ACE is more abundant in S3 than in S1 and S2 of PTCs, it would be important to determine the role of ACE in the S3 segment.

Background and Objectives: High-mobility group box 1 (HMGB1) is a highly conserved nonhistone DNA-binding nuclear protein which is present in almost all eukaryotic cells. HMGB1 is proposed to exert effects on several forms of vascular disease including atherosclerosis, pulmonary hypertension, and abdominal aortic aneurysm. However, the lack of a viable whole-body genetic deletion has increased interest in pharmacological approaches to sufficiently inhibit HMGB1 to investigate its role in pathological conditions. In the current study, we assessed the efficacy of a novel antisense oligonucleotides (ASOs) approach to deplete HMGB1 in mice. Methods and Results: Either ASOs (25 mg/kg/day) or phosphate-buffered saline (PBS) were injected intraperitoneally into male C57BL/6J mice (8-10-week-old). ASOs were injected at day 0 and day 3 in the initial week and once a week for the duration of the study. Mice were terminated at either 2, 4, or 12 weeks after the initial injection of ASOs (N = 5/group). Since HMGB1 is highly abundant in kidney and liver, mRNA and protein abundance of HMGB1 were examined subsequently in these organs by qPCR and Western blot analyses.ASO administration resulted in 90% decrease of Hmgb1 mRNA abundance in kidney and liver by 2 weeks of ASO injection. The decreased Hmbg1 mRNA abundance was observed consistently at both week 4 and 12. Surprisingly, ASOs failed to deplete HMGB1 protein abundance in kidney and liver at 2- and 4-week time intervals. However, HMGB1 protein abundance was markedly reduced to less than 20% in kidney and liver tissues at week 12, indicating an extended protein half-life. Conclusion: These findings reveal the protracted interval need to deplete HMGB1 protein depletion after mRNA inhibition.

Objective— AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results— AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor −/− mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions— These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.

Angiotensin II (AngII), a major effector of the renin-angiotensin system, exerts critical roles in regulating vascular function. AngII infusion induces abdominal aortic aneurysms (AAAs) and exacerbates atherosclerosis in hypercholesterolemic mice. We determined the effects of AngII infusion on endogenous AngII regulation and AngII-mediated AAAs and atherosclerosis. AngII infusion increased renal, but not plasma, AngII concentrations in male mice. AngI concentrations were decreased modestly in kidney, but more profoundly in plasma, during AngII infusion. Bovine AngII (DRVYVHPF) has one amino acid difference from mouse AngII (DRVYIHPF) that can be distinguished by LC-MS/MS. Therefore, we determined exogenous versus endogenous peptides in mice infused with bovine AngII. Bovine AngII infusion reduced endogenous renal AngII concentrations. To determine whether the residual endogenous AngII exerted an effect on AAAs and atherosclerosis in mice infused with AngII, aliskiren (a direct renin inhibitor) was administered to AngII-infused male LDL receptor deficient mice. Although aliskiren did not attenuate AAAs in AngII-infused mice, atherosclerotic lesion size was reduced. In conclusion, endogenous AngII concentrations are reduced during AngII infusion but still contribute to atherosclerosis, but not AAA, in AngII-infused hypercholesterolemic mice.

Objective: Cytoskeletal structural proteins maintain cell structural integrity by bridging extracellular matrix with contractile filaments. During abdominal aortic aneurysm (AAA) development, (1) aortic medial degeneration is associated with loss of smooth muscle cell integrity and (2) fibrogenic mesenchymal cells mediate extracellular matrix remodeling. Calpains cleave cytoskeletal proteins that maintain cell structural integrity. Pharmacological inhibition of calpains exert beneficial effects on Ang II (angiotensin II)–induced AAAs in LDLR −/− (low-density receptor deficient) mice. Here, we evaluated the functional contribution of fibrogenic mesenchymal cells-derived calpain-2 on (1) cytoskeletal structural protein and extracellular matrix alterations and (2) AAA progression. Approach and Results: Calpain-2 protein and cytoskeletal protein (filamin and talin) fragmentation are significantly elevated in human and Ang II–induced AAAs in mice. To examine the relative contribution of calpain-2 in AAA development, calpain-2 floxed mice in an LDLR −/− background were bred to mice with a tamoxifen-inducible form of Cre under control of either the ubiquitous promoter, chicken β-actin, or fibrogenic mesenchymal cell-specific promoter, Col1α2 (collagen type 1 alpha 2). Ubiquitous or fibrogenic mesenchymal cell-specific depletion of calpain-2 in mice suppressed Ang II–induced AAAs, filamin/talin fragmentation, while promoting extracellular matrix protein, collagen in the aortas. Calpain-2 silencing in aortic smooth muscle cells or fibroblasts reduced Ang II–induced filamin fragmentation. In addition, silencing of filamin in aortic SMCs significantly reduced collagen protein. Furthermore, calpain-2 deficiency suppressed rupture of established Ang II–induced AAAs in mice. Conclusions: Our studies implicate that calpain-2 deficiency prevents (1) Ang II–induced cytoskeletal structural protein fragmentation and AAA development and (2) stabilize and suppress rupture of established AAAs in mice.

ObjectiveAortic ruptures are fatal consequences of aortic aneurysms with macrophage accumulation being a hallmark at the site of ruptures. Pyroptosis is critical in macrophage-mediated inflammation. This study determined effects of pyroptosis on aortic dilation and rupture using GSDMD deficient mice.Approach and ResultsIn an initial study, male Gsdmd+/+ and Gsdmd-/- mice in C57BL/6J background (8 – 10 weeks old) were infected with adeno-associated viral vectors encoding mouse PCSK9D377Y gain-of-function mutation and fed a Western diet to induce hypercholesterolemia. After two weeks of AAV infection, angiotensin II (AngII, 1 µg/kg/min) was infused. During the 4 weeks of AngII infusion, 5 of 13 Gsdmd+/+ mice died of aortic rupture, whereas no aortic rupture occurred in Gsdmd-/- mice. In surviving mice, no differences in either ascending or abdominal aortic dilation were observed between Gsdmd+/+ and Gsdmd-/- mice. To determine whether protection of GSDMD deficiency against aortic rupture is specific to AngII infusion, we subsequently examined aortic pathologies in mice administered beta-aminopropionitrile (BAPN). BAPN (0.5% wt/vol) was administered in drinking water to male Gsdmd+/+ and Gsdmd-/- mice (4 weeks old) for 4 weeks. Six of 13 Gsdmd+/+ mice died of aortic rupture, whereas no aortic rupture occurred in Gsdmd-/- mice. In mice survived, no differences of diameters in the ascending, arch, or abdominal aortic regions were observed between Gsdmd+/+ and Gsdmd-/- mice.ConclusionsGSDMD deficiency protects against AngII or BAPN-induced aortic ruptures in mice.HighlightsGSDMD deficiency protects against angiotensin II-induced aortic rupture in hypercholesterolemic mice.GSDMD deficiency protects against beta-aminopropionitrile (BAPN)-induced aortic dissection and rupture in C57BL/6J mice.

Abscisic acid (ABA) and gibberellin (GA) are two antagonistic phytohormones that regulate seed germination in response to biotic and abiotic environmental stresses. We demonstrate here that MOTHER OF FT AND TFL1 (MFT), which encodes a phosphatidylethanolamine-binding protein, regulates seed germination via the ABA and GA signaling pathways in Arabidopsis thaliana. MFT is specifically induced in the radical-hypocotyl transition zone of the embryo in response to ABA, and mft loss-of-function mutants show hypersensitivity to ABA in seed germination. In germinating seeds, MFT expression is directly regulated by ABA-INSENSITIVE3 (ABI3) and ABI5, two key transcription factors in ABA signaling pathway. MFT is also upregulated by DELLA proteins in the GA signaling pathway. MFT in turn provides negative feedback regulation of ABA signaling by directly repressing ABI5. We conclude that during seed germination, MFT promotes embryo growth by constituting a negative feedback loop in the ABA signaling pathway.

Objective: Adrenal gland secretes stress-induced glucocorticoids (iGCs) to coping with stress. Previous study showed that SR-BI (scavenger receptor BI) null (SR-BI −/− ) mice failed to generate iGC in stress conditions, suggesting that SR-BI–mediated cholesterol uptake from HDL (high-density lipoprotein) is a key regulator for iGC production. However, the LDL (low-density lipoprotein)/LDLr (LDL receptor) pathway can also provide cholesterol for iGC synthesis, but rodents have limited LDL levels in circulation. Here, we generated SR-BI −/− ApoBtg (apolipoprotein B transgenic) mice with normal LDL levels in circulation to determine the relative contribution of the HDL/SR-BI and LDL/LDLr pathways to iGC production in stress conditions. Approach and Results: To obtain mouse models with normal LDL levels, SR-BI −/− mice were bred to ApoBtg mice. Then, the F1 SR-BI±ApoBtg mice were backcrossed to SR-BI −/− to obtain SR-BI −/− ApoBtg, SR-BI −/− ApoBwt (apolipoprotein B wild type), and SR-BI +/+ ApoBtg mice. We first examined the lipoprotein profile, which shows a 6.5-fold increase in LDL levels in SR-BI −/− ApoBtg mice compared with SR-BI −/− ApoBwt mice. Then, we induced stress with adrenocorticotropic hormone and cecal ligation and puncture. One hour after adrenocorticotropic hormone stimulation, SR-BI +/+ ApoBtg control mice produced iGC (14.9-fold), but both SR-BI −/− ApoBwt and SR-BI −/− ApoBtg showed no iGC production ( P +/+ ApoBtg control mice showed iGC production (6.4-fold), but both SR-BI −/− ApoBwt and SR-BI −/− ApoBtg mice showed no iGC production ( P Conclusions: SR-BI −/− ApoBtg mice fail to produce iGC in stress conditions even though with restored LDL levels in circulation. These findings clarify that the HDL/SR-BI, not LDL/LDLr, pathway is responsible for iGC production in stress conditions.

Graphic AbstractObjectiveA cardinal feature of Marfan syndrome is thoracic aortic aneurysm (TAA). The contribution of ligand-dependent stimulation of angiotensin II receptor type 1a (AT1aR) to TAA progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacologic modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on TAA in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+).Approach and ResultsTAA in Fbn1C1041G/+ mice were determined in both sexes and found to be strikingly sexual dimorphic. Males displayed progressive dilation over 12 months while ascending aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or −/− for AT1aR were generated. AT1aR deletion reduced progressive expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen (AGT-ASO) was administered to male Fbn1C1041G/+ mice to determine the effects of angiotensin II depletion. AGT-ASO administration, at doses that markedly reduced plasma AGT concentrations, attenuated progressive dilation of the ascending aorta and aortic root. AGT-ASO also reduced medial thickening and elastin fragmentation.ConclusionsGenetic approaches to delete AT1aR and deplete AngII production exerted similar effects in attenuating pathology in the proximal thoracic aorta of male Fbn1C1041G/+ mice. These data are consistent with ligand (AngII) dependent stimulation of AT1aR being responsible for aortic disease progression.HighlightsProfound sexual dimorphism of aortic disease occurs in Fbn1C1041G/+ mice, with female mice being more resistant and male mice being more susceptible.Inhibition of the AngII-AT1aR axis attenuates aortic pathology in male Fbn1C1041G/+ mice.Antisense oligonucleotides targeting angiotensinogen deplete plasma angiotensinogen and attenuate thoracic aortic aneurysms.

OBJECTIVE: This study determined whether hypercholesterolemia would contribute to both the initiation and progression of angiotensin (Ang)II-induced abdominal aortic aneurysms (AAAs) in mice. METHODS AND RESULTS: To determine whether hypercholesterolemia accelerates the initiation of AAAs, male low-density lipoprotein (LDL) receptor -/- mice were either fed one week of Western diet prior to starting AngII infusion or initiated Western diet one week after starting AngII infusion. During the first week of AngII infusion, mice fed normal diet had less luminal expansion of the suprarenal aorta compared to those initiated Western diet after the first week of AngII infusion. The two groups achieved comparable luminal dilation on week 2 through week 6 of AngII infusion as monitored by ultrasound. To determine whether hypercholesterolemia contributed to the progression of established AAAs, male LDL receptor -/- mice were fed Western diet and infused with AngII for 4 weeks. Mice with established AAAs were then stratified into two groups based on luminal diameters measured by ultrasound. While AngII infusion was continued for another 8 weeks in both groups, mice in one group were continuously fed Western diet, but diet in the other group was switched to normal laboratory diet. In the latter group, plasma cholesterol concentrations were reduced rapidly to approximately 500 mg/dl within one week after the diet was switched from Western diet to normal laboratory diet. Luminal expansion progressed constantly in mice continuously fed Western diet, whereas no continuous expansion was detected in mice that were switched to normal laboratory diet. CONCLUSION: Hypercholesterolemia accelerates both the initiation of AAAs and progression of established AAAs in AngII-infused male LDL receptor -/- mice. CLINICAL RELEVANCE: Hypercholesterolemia is modestly associated with AAAs in observational or retrospective clinical studies. It is not feasible to study whether hypercholesterolemia contributes to the initiation of AAAs or progression of established AAAs in human. This study using AngII-induced AAA mouse model provides solid evidence that hypercholesterolemia contributes to both the initiation and progression of AAAs, supporting that statin therapy at any stage of AAA development may be beneficial to hypercholesterolemic patients with AAAs.

Momordica Charantia, commonly known as bitter melon, has been reported to ameliorate diet-induced obesity and dyslipidemia. However, the effects of M. Charantia on atherosclerosis have not been determined. This study investigated the effects of M. Charantia on diet-induced atherosclerosis in LDL receptor deficient mice. Female mice (6-8 weeks old) were fed a saturated fat-enriched diet. In group 1, mice were fed this diet alone, while mice in groups 2 and 3 were fed the diet supplemented with M. Charantia either 0.1% or 1% by weight, for 12 weeks. No significant differences in body weights were observed among the 3 groups. There were also no significant differences in plasma cholesterol concentrations among the 3 groups. To determine the effects on atherosclerosis, lesion areas were measured on aortic intima by an en face technique. Neither dose of M. Charantia supplementation changed atherosclerosis development.

This protocol is used for generation of ascending aortic serial sections for histology. This protocol generates a set of 10 slides covering ~1mm of ascending aortic tissue. Inclusion of the descening aorta provides orientation and a non-TAA control from the same animal.

Disruption of the circadian system caused by disordered exposure to light is pervasive in modern society and increases the risk of cardiovascular disease. The mechanisms by which this happens are largely unknown. ApolipoproteinE-deficient (ApoE−/−) mice are studied commonly to elucidate mechanisms of atherosclerosis. In this study, we determined the effects of light-induced circadian disruption on atherosclerosis in ApoE−/− mice. We first characterized circadian rhythms of behavior, light responsiveness, and molecular timekeeping in tissues from ApoE−/− mice that were indistinguishable from rhythms in ApoE+/+ mice. These data showed that ApoE−/− mice had no inherent circadian disruption and therefore were an appropriate model for our study. We next induced severe disruption of circadian rhythms by exposing ApoE−/− mice to constant light for 12 weeks. Constant light exposure exacerbated atherosclerosis in male, but not female, ApoE−/− mice. Male ApoE−/− mice exposed to constant light had increased serum cholesterol concentrations due to increased VLDL/LDL fractions. Taken together, these data suggest that ApoE−/− mice are an appropriate model for studying light-induced circadian disruption and that exacerbated dyslipidemia may mediate atherosclerotic lesion formation caused by constant light exposure.

BackgroundThe ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies.MethodsAscending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry assisted proteomics, and molecular features of SHF-derived cells were determined by single cell transcriptomic analyses. Genetic deletion of either low-density lipoprotein receptor-related protein 1 (Lrp1) or transforming growth factor-β receptor 2 (Tgfbr2) in SHF- derived cells was conducted to examine the impact of SHF-derived cells on vascular integrity.ResultsPathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas following AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion, prior to overt pathology, evoked downregulation of SMC proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single cell transcriptomics results identified plasminogen activator inhibitor 1 (PAI1) as the most increased protein in SHF-derived SMCs and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies.ConclusionSHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and TGF-β signaling associated with increases of aortic PAI1.Abstract FigureClinical PerspectiveWhat is new?-SHF-derived SMCs and fibroblasts associate with AngII-induced aortic pathologies.-AngII induces a distinct fibroblast sub-cluster that is less abundant for mRNAs related to major extracellular components and TGFβ ligands and receptors, but more abundant for proliferative genes.-TGFBR2 deletion in SHF-derived cells are embryonic lethal with significant dilatation of the outflow tract in mice.-SHF-specific deletion of LRP1 leads to aortic pathologies in mice, supporting the importance of SHF-derived cells in maintaining ascending aortic wall integrity.What are the clinical implications?-Heterogeneity of the embryonic origins of SMCs and fibroblasts contributes to complex mechanisms of vasculopathy formation, which should be considered when investigating the pathogenesis of thoracic aortopathies.

ObjectiveThis study determined whether hypercholesterolemia would contribute to both the initiation and progression of angiotensin (Ang)II-induced abdominal aortic aneurysms (AAAs) in mice.Methods and ResultsTo determine whether hypercholesterolemia accelerates the initiation of AAAs, male low-density lipoprotein (LDL) receptor -/- mice were either fed one week of Western diet prior to starting AngII infusion or initiated Western diet one week after starting AngII infusion. During the first week of AngII infusion, mice fed normal diet had less luminal expansion of the suprarenal aorta compared to those initiated Western diet after the first week of AngII infusion. The two groups achieved comparable luminal dilation on week 2 through week 6 of AngII infusion as monitored by ultrasound. To determine whether hypercholesterolemia contributed to the progression of established AAAs, male LDL receptor -/- mice were fed Western diet and infused with AngII for 4 weeks. Mice with established AAAs were then stratified into two groups based on luminal diameters measured by ultrasound. While AngII infusion was continued for another 8 weeks in both groups, mice in one group were continuously fed Western diet, but diet in the other group was switched to normal laboratory diet. In the latter group, plasma cholesterol concentrations were reduced rapidly to approximately 500 mg/dl within one week after the diet was switched from Western diet to normal laboratory diet. Luminal expansion progressed constantly in mice continuously fed Western diet, whereas no continuous expansion was detected in mice that were switched to normal laboratory diet.ConclusionsHypercholesterolemia accelerates both the initiation of AAAs and progression of established AAAs in AngII-infused male LDL receptor -/- mice.Clinical RelevanceHypercholesterolemia is modestly associated with AAAs in observational or retrospective clinical studies. It is not feasible to study whether hypercholesterolemia contributes to the initiation of AAAs or progression of established AAAs in human. This study using AngII-induced AAA mouse model provides solid evidence that hypercholesterolemia contributes to both the initiation and progression of AAAs, supporting that statin therapy at any stage of AAA development may be beneficial to hypercholesterolemic patients with AAAs.

Objective— Continuous T-cell production from thymus is essential in replenishing naïve T-cell pool and maintaining optimal T-cell functions. However, the underlying mechanisms regulating the T-cell development in thymus remains largely unknown. Approach and Results— We identified SR-BI (scavenger receptor class B type 1), an HDL (high-density lipoprotein) receptor, as a novel modulator in T-cell development. We found that SR-BI deficiency in mice led to reduced thymus size and decreased T-cell production, which was accompanied by narrowed peripheral naïve T-cell pool. Further investigation revealed that SR-BI deficiency impaired progenitor thymic homing, causing a dramatic reduction in the percentage of earliest thymic progenitors, but did not affect other downstream T-cell developmental steps inside the thymus. As a result of the impaired progenitor thymic homing, SR-BI-deficient mice displayed delayed thymic regeneration postirradiation. Using a variety of experimental approaches, we revealed that the impaired T-cell development in SR-BI-deficient mice was not caused by hematopoietic SR-BI deficiency or SR-BI deficiency-induced hypercholesterolemia, but mainly attributed to the SR-BI deficiency in adrenal glands, as adrenal-specific SR-BI-deficient mice exhibited similar defects in T-cell development and thymic regeneration with SR-BI-deficient mice. Conclusions— This study demonstrates that SR-BI deficiency impaired T-cell development and delayed thymic regeneration by affecting progenitor thymic homing in mice, elucidating a previously unrecognized link between SR-BI and adaptive immunity.

ObjectiveRenin cleavage of angiotensinogen (AGT) has species specificity. Since the residues at positions 11 and 12 of AGT are different between human and mouse AGT, we determined whether these two residues in AGT affect renin cleavage and angiotensin II-mediated functions using an adeno-associated viral (AAV) approach for manipulating AGT in vivo.Approach and ResultsHepatocyte-specific AGT deficient (hepAGT-/-) mice in an LDL receptor -/- background were infected with AAVs containing a null insert, human AGT, or mouse AGT expressing the same residues of the human protein at positions 11 and 12 [mouse AGT (L11V;Y12I)]. Expression of human AGT in hepAGT-/- mice led to high plasma human AGT concentrations without changes in plasma mouse endogenous AGT, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human AGT not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human AGT lead to the inability of mouse renin to cleave human AGT, hepAGT-/- mice were injected with AAV encoding mouse AGT (L11V;Y12I). Expression of mouse AGT (L11V;Y12I) resulted in increased plasma mouse AGT concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse AGT variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice.ConclusionReplacement of L11 and Y12 to V11 and I12, respectively, in mouse AGT does not affect renin cleavage and AngII-mediated functions in mice.HIGHLIGHTSHuman AGT is not cleaved by mouse renin and does not change AngII-mediated functions in hepatocyte-specific AGT -/- mice.Replacement of the N-terminal amino acids at 11 and 12 positions from mouse to human AGT does not affect renin cleavage and AngII-mediated functions in hepatocyte-specific AGT -/- mice.

Objective— Angiotensin-converting enzyme (ACE) is present in many cell types of atherosclerotic lesions. This study determined whether ACE activity in endothelial and smooth muscle cells (SMCs), 2 major resident cell types of the aorta, contributes to hypercholesterolemia-induced atherosclerosis. Approach and Results— All study mice were in low-density lipoprotein receptor −/− background. To determine the contribution of ACE on endothelial cells to atherosclerosis, female ACE floxed mice were bred to male Tie2-Cre transgenic mice. Endothelial cell–specific deletion of ACE significantly decreased serum ACE activity, but had no effect on systolic blood pressure and atherosclerosis. Because ACE protein is present on SMCs, the most abundant cell type of the aorta, we then determined whether ACE on SMCs contributes to atherosclerosis. ACE was depleted from SMCs by breeding female ACE floxed mice with male SM22-Cre transgenic mice. SMC-specific deficiency of ACE did not affect ACE activity in serum, but ablated its presence and activity in the aortic media. Although SMC-specific deficiency of ACE had no effect on systolic blood pressure, it significantly attenuated hypercholesterolemia-induced atherosclerosis in both male and female mice. Conclusions— These studies provide direct evidence that ACE derived from endothelial cells does not play a critical role in atherosclerosis. Rather, SMC-derived ACE contributes to atherosclerosis, independent of circulating ACE activity and blood pressure.

Objective— Angiotensin II (AngII) infusion profoundly increases activity of calpains, calcium-dependent neutral cysteine proteases, in mice. Pharmacological inhibition of calpains attenuates AngII-induced aortic medial macrophage accumulation, atherosclerosis, and abdominal aortic aneurysm in mice. However, the precise functional contribution of leukocyte-derived calpains in AngII-induced vascular pathologies has not been determined. The purpose of this study was to determine whether calpains expressed in bone marrow (BM)–derived cells contribute to AngII-induced atherosclerosis and aortic aneurysms in hypercholesterolemic mice. Approach and Results— To study whether leukocyte calpains contributed to AngII-induced aortic pathologies, irradiated male low-density lipoprotein receptor −/− mice were repopulated with BM-derived cells that were either wild-type or overexpressed calpastatin, the endogenous inhibitor of calpains. Mice were fed a fat-enriched diet and infused with AngII (1000 ng/kg per minute) for 4 weeks. Overexpression of calpastatin in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches, but had no effect on aneurysm formation. Using either BM-derived cells from calpain-1-deficient mice or mice with leukocyte-specific calpain-2 deficiency generated using cre-loxP recombination technology, further studies demonstrated that independent deficiency of either calpain-1 or -2 in leukocytes modestly attenuated AngII-induced atherosclerosis. Calpastatin overexpression significantly attenuated AngII-induced inflammatory responses in macrophages and spleen. Furthermore, calpain inhibition suppressed migration and adhesion of macrophages to endothelial cells in vitro. Calpain inhibition also significantly decreased hypercholesterolemia-induced atherosclerosis in the absence of AngII. Conclusions— The present study demonstrates a pivotal role for BM-derived calpains in mediating AngII-induced atherosclerosis by influencing macrophage function.

Objective— This study determined whether angiotensinogen (AGT) has angiotensin II–independent effects using multiple genetic and pharmacological manipulations. Approach and Results— All study mice were in low-density lipoprotein receptor −/− background and fed a saturated fat–enriched diet. In mice with floxed alleles and a neomycin cassette in intron 2 of the AGT gene (hypoAGT mice), plasma AGT concentrations were >90% lower compared with their wild-type littermates. HypoAGT mice had lower systolic blood pressure, less atherosclerosis, and diminished body weight gain and liver steatosis. Low plasma AGT concentrations and all phenotypes were recapitulated in mice with hepatocyte-specific deficiency of AGT or pharmacological inhibition of AGT by antisense oligonucleotide administration. In contrast, inhibition of AGT cleavage by a renin inhibitor, aliskiren, failed to alter body weight gain and liver steatosis in low-density lipoprotein receptor −/− mice. In mice with established adiposity, administration of AGT antisense oligonucleotide versus aliskiren led to equivalent reductions of systolic blood pressure and atherosclerosis. AGT antisense oligonucleotide administration ceased body weight gain and further reduced body weight, whereas aliskiren did not affect body weight gain during continuous saturated fat–enriched diet feeding. Structural comparisons of AGT proteins in zebrafish, mouse, rat, and human revealed 4 highly conserved sequences within the des(angiotensin I)AGT domain. des(angiotensin I)AGT, through adeno-associated viral infection in hepatocyte-specific AGT-deficient mice, increased body weight gain and liver steatosis, but did not affect atherosclerosis. Conclusions— AGT contributes to body weight gain and liver steatosis through functions of the des(angiotensin I)AGT domain, which are independent of angiotensin II production.

Bitter melon (BM; Momordica charantia L.) has been reported to ameliorate diet-induced obesity and dyslipidemia. However, the effects of BM on atherosclerosis have not been determined. This study investigated the effects of BM diet-induced atherosclerosis in LDL receptor-deficient mice. A total of 30 female mice (aged 6-8 wk) were fed a saturated fat-enriched diet. In group 1 (n = 10), mice were fed this diet alone, whereas mice in groups 2 and 3 (n = 10/group) were fed the diet supplemented with BM either 0.1% or 1% by weight. After 12 wk, body weight, plasma cholesterol, and atherosclerotic plaque areas were analyzed. No significant differences in body weight and plasma cholesterol concentrations were observed among the groups. Also, BM supplementation did not affect atherosclerosis development. In conclusion, dietary BM has no effect on plasma cholesterol concentration and atherogenesis in hypercholesterolemic mice.

Background and objective: Our previous study demonstrated that deficiency of toll-like receptor 4 (TLR4) reduced angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Lipopolysaccharide (LPS) is a well-known agonist of TLR4. Interaction of LPS and TLR4 promotes inflammation and augments hypercholesterolemia-induced atherosclerosis. The purpose of this study was to determine whether LPS augments AngII-induced AAAs. Methods and Results: Low-density lipoprotein (LDL) receptor -/- mice fed a Western diet develop hypercholesterolemia and have augmentation of AngII-induced AAAs. We found that LDL receptor -/- mice fed Western diet had increased LPS concentrations, as quantified by a Limulus Amebocyte lysate chromogenic endotoxin quantitation kit, compared to the same mice prior to Western diet feeding. To determine whether LPS plays a role on AngII-induced AAAs, 4 groups of male C57BL/6J mice, 10-week-old, were infused subcutaneously with: (1) Vehicle (N=5), (2) LPS (250 μ g/kg/day; N=10), (3) AngII (1,000 ng/kg/min; N=10), or (4) both AngII and LPS (N=10) through Alzet mini osmotic pumps for 28 days. Abdominal aortic expansion was monitored serially at 2 and 4 weeks of infusion using high frequency ultrasonography (Vevo 3100 system; FUJIFILM). Continuous luminal expansion was detected in both AngII-infused mice and mice co-infused with both AngII and LPS. These results were confirmed by ex vivo measurements of maximal outer diameters of suprarenal aortas after termination showing AngII infusion led to significant increase of abdominal aortic dilation, compared to vehicle or LPS infused mice. However, co-infusion of LPS with AngII did not augment AngII-induced AAAs. Conclusion: LPS has no effects on AngII-induced AAAs in normolipidemic mice.