ß-Aminopropionitrile Induces Distinct Pathologies in the Ascending and Descending Thoracic Aortic Regions of Mice.

BACKGROUND: ß-aminopropionitrile (BAPN) is a pharmacological inhibitor of LOX (lysyl oxidase) and LOXLs (LOX-like proteins). Administration of BAPN promotes aortopathies, although there is a paucity of data on experimental conditions to generate pathology. The objective of this study was to define experimental parameters and determine whether equivalent or variable aortopathies were generated throughout the aortic tree during BAPN administration in mice. METHODS: BAPN was administered in drinking water for a period ranging from 1 to 12 weeks. The impacts of BAPN were first assessed with regard to dose, strain, age, and sex. BAPN-induced aortic pathological characterization was conducted using histology and immunostaining. To investigate the mechanistic basis of regional heterogeneity, the ascending and descending thoracic aortas were harvested after 1 week of BAPN administration before the appearance of overt pathology. RESULTS: BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta in young C57BL/6J or N mice. No apparent differences were found between male and female mice. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-SMA (α-smooth muscle actin). One week of BAPN administration compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the 2 aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components. CONCLUSIONS: BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in mice.

Renal Proximal Tubule Cell-specific Megalin Deletion Induces Tubulointerstitial Nephritis in Mice Fed Western Diet.

Pharmacological inhibition of megalin (also known as low-density lipoprotein receptor-related protein 2: LRP2) attenuates atherosclerosis in hypercholesterolemic mice. Since megalin is abundant in renal proximal tubule cells (PTCs), PTC-LRP2 +/+ and -/- littermates in an LDL receptor -/- background were generated and fed a Western diet to determine effects of PTC-derived megalin on atherosclerosis. PTC-specific megalin deletion did not attenuate atherosclerosis in LDL receptor -/- mice in either sex. Serendipitously, we discovered that PTC-specific megalin deletion led to interstitial infiltration of CD68+ cells and tubular atrophy. The pathology was only evident in male PTC-LRP2 -/- mice fed the Western diet, but not in mice fed a normal laboratory diet. Renal pathologies were also observed in male PTC-LRP2 -/- mice in an LDL receptor +/+ background fed the same Western diet, demonstrating that the renal pathologies were dependent on diet and not hypercholesterolemia. By contrast, female PTC-LRP2 -/- mice had no apparent renal pathologies. In vivo multiphoton microscopy demonstrated that PTC-specific megalin deletion dramatically diminished albumin accumulation in PTCs within 10 days of Western diet feeding. RNA sequencing analyses demonstrated the upregulation of inflammation-related pathways in kidney. Overall, PTC-specific megalin deletion leads to tubulointerstitial nephritis in mice fed Western diet, with severe pathologies in male mice.

Angiotensinogen as a Therapeutic Target for Cardiovascular and Metabolic Diseases.

AGT (angiotensinogen) is the unique precursor for the generation of all the peptides of the renin-angiotensin system, but it has received relatively scant attention compared to many other renin-angiotensin system components. Focus on AGT has increased recently, particularly with the evolution of drugs to target the synthesis of the protein. AGT is a noninhibitory serpin that has several conserved domains in addition to the angiotensin II sequences at the N terminus. Increased study is needed on the structure-function relationship to resolve many unknowns regarding AGT metabolism. Constitutive whole-body genetic deletion of Agt in mice leads to multiple developmental defects creating a challenge to use these mice for mechanistic studies. This has been overcome by creating Agt-floxed mice to enable the development of cell-specific deficiencies that have provided considerable insight into a range of cardiovascular and associated diseases. This has been augmented by the recent development of pharmacological approaches targeting hepatocytes in humans to promote protracted inhibition of AGT synthesis. Genetic deletion or pharmacological inhibition of Agt has been demonstrated to be beneficial in a spectrum of diseases experimentally, including hypertension, atherosclerosis, aortic and superior mesenteric artery aneurysms, myocardial dysfunction, and hepatic steatosis. This review summarizes the findings of recent studies utilizing AGT manipulation as a therapeutic approach.

Association of NOTCH3 With Elastic Fiber Dispersion in the Infrarenal Abdominal Aorta of Cynomolgus Monkeys.

BACKGROUND: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in nonhuman primates. METHODS: Aortic samples were harvested from the ascending, descending thoracic, suprarenal, and infrarenal regions of young control monkeys and adult monkeys with high fructose consumption for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses, respectively. RESULTS: Immunostaining of CD31 and αSMA (alpha-smooth muscle actin) revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared with other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared with other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys with high fructose consumption displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. CONCLUSIONS: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3.

ß-aminopropionitrile Induces Distinct Pathologies in the Ascending and Descending Thoracic Aortic Regions of Young Mice.

This study characterized ß-aminopropionitrile (BAPN)-induced aortopathies in young mice. The effects of BAPN were first determined with regard to BAPN dose and mouse strain, age, and sex. BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, macrophage infiltration, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-smooth muscle actin. To investigate the molecular basis of the regional heterogeneity, ascending and descending thoracic aortas were harvested after one week of BAPN administration prior to the appearance of overt pathology. BAPN compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the two aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components. In conclusion, BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in young mice.

Manipulation of components of the renin angiotensin system in renal proximal tubules fails to alter atherosclerosis in hypercholesterolemic mice.

Background and objective: Whole body manipulation of the renin-angiotensin system (RAS) consistently exerts profound effects on experimental atherosclerosis development. A deficit in the literature has been a lack of attention to the effects of sex. Also, based on data with gene-deleted mice, the site of RAS activity that influences lesion formation is at an unknown distant location. Since angiotensin (AngII) concentrations are high in kidney and the major components of the RAS are present in renal proximal tubule cells (PTCs), this study evaluated the role of the RAS in PTCs in atherosclerosis development. Methods and results: Mice with an LDL receptor -/- background were fed Western diet to induce hypercholesterolemia and atherosclerosis. We first demonstrated the role of AT1 receptor antagonism on atherosclerosis in both sexes. Losartan, an AngII type 1 (AT1) receptor blocker, had greater blood pressure-lowering effects in females than males, but equivalent effects between sexes in reducing atherosclerotic lesion size. To determine the roles of renal AT1a receptor and angiotensin-converting enzyme (ACE), either component was deleted in PTCs after weaning using a tamoxifen-inducible Cre expressed under the control of an Ndrg1 promoter. Despite profound deletion of AT1a receptor or ACE in PTCs, the absence of either protein did not influence development of atherosclerosis in either sex. Conversely, mice expressing human angiotensinogen and renin in PTCs or expressing human angiotensinogen in liver but human renin in PTCs did not change atherosclerotic lesion size in male mice. Conclusion: Whole-body AT1R inhibition reduced atherosclerosis equivalently in both male and female mice; however, PTC-specific manipulation of the RAS components had no effects on hypercholesterolemia-induced atherosclerosis.

Association of NOTCH3 with Elastic Fiber Dispersion in the Infrarenal Abdominal Aorta of Cynomolgus Monkeys.

Background: The regional heterogeneity of vascular components and transcriptomes is an important determinant of aortic biology. This notion has been explored in multiple mouse studies. In the present study, we examined the regional heterogeneity of aortas in non-human primates. Methods: Aortic samples were harvested from the ascending, descending, suprarenal, and infrarenal regions of young control monkeys and adult monkeys provided with high fructose for 3 years. The regional heterogeneity of aortic structure and transcriptomes was examined by histological and bulk RNA sequencing analyses. Results: Immunostaining of CD31 and αSMA revealed that endothelial and smooth muscle cells were distributed homogeneously across the aortic regions. In contrast, elastic fibers were less abundant and dispersed in the infrarenal aorta compared to other regions and associated with collagen deposition. Bulk RNA sequencing identified a distinct transcriptome related to the Notch signaling pathway in the infrarenal aorta with significantly increased NOTCH3 mRNA compared to other regions. Immunostaining revealed that NOTCH3 protein was increased in the media of the infrarenal aorta. The abundance of medial NOTCH3 was positively correlated with the dispersion of elastic fibers. Adult cynomolgus monkeys provided with high fructose displayed vascular wall remodeling, such as smooth muscle cell loss and elastic fiber disruption, predominantly in the infrarenal region. The correlation between NOTCH3 and elastic fiber dispersion was enhanced in these monkeys. Conclusions: Aortas of young cynomolgus monkeys display regional heterogeneity of their transcriptome and the structure of elastin and collagens. Elastic fibers in the infrarenal aorta are dispersed along with upregulation of medial NOTCH3. HIGHLIGHTS: - The present study determined the regional heterogeneity of aortas from cynomolgus monkeys.- Aortas of young cynomolgus monkeys displayed region-specific aortic structure and transcriptomes.- Elastic fibers were dispersed in the infrarenal aorta along with increased NOTCH3 abundance in the media.

Recent Advances in Understanding the Molecular Pathophysiology of Angiotensin II Receptors: Lessons From Cell-Selective Receptor Deletion in Mice.

The renin-angiotensin system (RAS) is an essential hormonal system involved in water and sodium reabsorption, renal blood flow regulation, and arterial constriction. Systemic stimulation of the RAS with infusion of the main peptide angiotensin II (Ang II) in animals as well as pathological elevation of renin (ie, renovascular hypertension) to increase circulatory Ang II in humans ultimately lead to hypertension and end organ damage. In addition to hypertension, accumulating evidence supports that the Ang II type 1 receptor exerts a critical role in cardiovascular and kidney diseases independent of blood pressure elevation. In the past 2 decades, the identification of an increased number of peptides and receptors has facilitated the concept that the RAS has detrimental and beneficial effects on the cardiovascular system depending on which RAS components are activated. For example, angiotensin 1-7 and Ang II type 2 receptors act as a counter-regulatory system against the classical RAS by mediating vasodilation. Although the RAS as an endocrine system for regulation of blood pressure is well established, there remain many unanswered questions and controversial findings regarding blood pressure regulation and pathophysiological regulation of cardiovascular diseases at the tissue level. This review article includes the latest knowledge gleaned from cell type-selective gene deleted mice regarding cell type-specific roles of Ang II receptors and their significance in health and diseases are discussed. In particular, we focus on the roles of these receptors expressed in vascular, cardiac, and kidney epithelial cells.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient 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. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-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.

LRP1 protects against excessive superior mesenteric artery remodeling by modulating angiotensin II-mediated signaling.

Vascular smooth muscle cells (vSMCs) 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 signaling receptor that recognizes numerous ligands, including apolipoprotein E-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 vSMCs (smLRP1-/- mice). Quantitative proteomics revealed elevated abundance of several proteins in smLRP1-/- mice that are known to be induced by angiotensin II-mediated (AngII-mediated) signaling, suggesting that this pathway was 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, using 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.

Aortic Aneurysm and Dissection: Heterogeneity and Molecular Mechanisms.

Aortic aneurysms and dissections (AAD) are devastating aortic diseases with high risks for aortic rupture, leading to uncontrolled bleeding and death [...].

Embryonic Heterogeneity of Smooth Muscle Cells in the Complex Mechanisms of Thoracic Aortic Aneurysms.

Smooth muscle cells (SMCs) are the major cell type of the aortic wall and play a pivotal role in the pathophysiology of thoracic aortic aneurysms (TAAs). TAAs occur in a region-specific manner with the proximal region being a common location. In this region, SMCs are derived embryonically from either the cardiac neural crest or the second heart field. These cells of distinct origins reside in specific locations and exhibit different biological behaviors in the complex mechanism of TAAs. The purpose of this review is to enhance understanding of the embryonic heterogeneity of SMCs in the proximal thoracic aorta and their functions in TAAs.

A mini-review on quantification of atherosclerosis in hypercholesterolemic mice.

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.

Inhibition of the Renin-Angiotensin System Fails to Suppress ß-Aminopropionitrile-Induced Thoracic Aortopathy in Mice-Brief Report.

BACKGROUND: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (ß-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.

Expression of a PCSK9 Gain-of-Function Mutation in C57BL/6J Mice to Facilitate Angiotensin II-Induced AAAs.

Angiotensin II (AngII) infusion in mice has been used widely to investigate mechanisms of abdominal aortic aneurysms (AAAs). To achieve a high incidence of AngII-induced AAAs, mice should be hypercholesterolemic. Therefore, either low-density lipoprotein receptor (LDLR) or apolipoprotein E deficiency have been used as a hypercholesterolemic background. However, it is a time-consuming and expensive process to generate compound deficient strains that have either an LDLR or apolipoprotein E deficient background. Proprotein convertase subtilisin/kexin type 9 (PCSK9) facilitates the degradation of LDL receptors. Previous studies demonstrated profound increases of plasma cholesterol concentrations after a single intraperitoneal injection of adeno-associated viruses (AAV) expressing a gain-of-function mutation of mouse PCSK9 (AAV.mPCSK9D377Y) in C57BL/6J mice fed a Western diet. Of note, injection of AAV.mPCSK9D377Y augmented AngII-induced AAA formation in C57BL/6J mice that had comparable severity of AAAs to LDLR deficient mice. Thus, AAV.mPCSK9D377Y infection greatly expedites studies on a gene of interest using AngII-induced AAAs. This commentary provides a brief technical guide of this approach and discusses the pros and cons of its use in AAA research.

Fludrocortisone Induces Aortic Pathologies in Mice.

BACKGROUND AND OBJECTIVE: In an experiment designed to explore the mechanisms of fludrocortisone-induced high blood pressure, we serendipitously observed aortic aneurysms in mice infused with fludrocortisone. The purpose of this study was to investigate whether fludrocortisone induces aortic pathologies in both normocholesterolemic and hypercholesterolemic mice. METHODS AND RESULTS: Male adult C57BL/6J mice were infused with either vehicle (85% polyethylene glycol 400 (PEG-400) and 15% dimethyl sulfoxide (DMSO); n = 5) or fludrocortisone (12 mg/kg/day dissolved in 85% PEG-400 and 15% DMSO; n = 15) for 28 days. Fludrocortisone-infused mice had higher systolic blood pressure, compared to mice infused with vehicle. Fludrocortisone induced aortic pathologies in 4 of 15 mice with 3 having pathologies in the ascending and aortic arch regions and 1 having pathology in both the ascending and descending thoracic aorta. No pathologies were noted in abdominal aortas. Subsequently, we infused either vehicle (n = 5/group) or fludrocortisone (n = 15/group) into male ApoE -/- mice fed a normal laboratory diet or LDL receptor -/- mice fed either normal or Western diet. Fludrocortisone increased systolic blood pressure, irrespective of mouse strain or diet. In ApoE -/- mice infused with fludrocortisone, 2 of 15 mice had ascending aortic pathologies, but no mice had abdominal aortic pathologies. In LDL receptor -/- mice fed normal diet, 5 had ascending/arch pathologies and 1 had pathologies in the ascending, arch, and suprarenal aortic regions. In LDL receptor -/- mice fed Western diet, 2 died of aortic rupture in either the descending thoracic or abdominal region, and 2 of the 13 survived mice had ascending/arch aortic pathologies. Aortic pathologies included hemorrhage, wall thickening or thinning, or dilation. Only ascending aortic diameter in LDLR -/- mice fed Western diet reached statistical significance, compared to their vehicle. CONCLUSION: Fludrocortisone induces aortic pathologies independent of hypercholesterolemia. As indicated by the findings in mouse studies, people who are taking or have taken fludrocortisone might have an increased risk of aortic pathologies.