Proteoglycan accumulation is a hallmark of medial degeneration in thoracic aortic aneurysm and dissection (TAAD). Here, we defined the aortic proteoglycanome using mass spectrometry, and based on the findings, investigated the large aggregating proteoglycans aggrecan and versican in human ascending TAAD and a mouse model of severe Marfan syndrome. The aortic proteoglycanome comprises 20 proteoglycans including aggrecan and versican. Antibodies against these proteoglycans intensely stained medial degeneration lesions in TAAD, contrasting with modest intralamellar staining in controls. Aggrecan, but not versican, was increased in longitudinal analysis of Fbn1mgR/mgR aortas. TAAD and Fbn1mgR/mgR aortas had increased aggrecan and versican mRNAs, and reduced expression of a key proteoglycanase gene, ADAMTS5, was seen in TAAD. Fbn1mgR/mgR mice with ascending aortic dissection and/or rupture had dramatically increased aggrecan staining compared with mice without these complications. Thus, aggrecan and versican accumulation in ascending TAAD occurs via increased synthesis and/or reduced proteolytic turnover, and correlates with aortic dissection/rupture in Fbn1mgR/mgR mice. Tissue swelling imposed by aggrecan and versican is proposed to be profoundly deleterious to aortic wall mechanics and smooth muscle cell homeostasis, predisposing to type-A dissections. These proteoglycans provide potential biomarkers for refined risk stratification and timing of elective aortic aneurysm repair.
Frank S. Cikach, Christopher D. Koch, Timothy J. Mead, Josephine Galatioto, Belinda B. Willard, Kelly B. Emerton, Matthew J. Eagleton, Eugene H. Blackstone, Francesco Ramirez, Eric E. Roselli, Suneel S. Apte
Von Hippel-Lindau (VHL) gene mutations induce neural tissue hemangioblastomas, as well as highly vascularized clear cell renal cell carcinomas (ccRCCs). Pathological vessel remodeling arises from misregulation of HIFs and VEGF, among other genes. Variation in disease penetrance has long been recognized in relation to genotype. We show Vhl mutations also disrupt Notch signaling, causing mutation-specific vascular abnormalities, e.g., type 1 (null) vs. type 2B (murine G518A representing human R167Q). In conditional mutation retina vasculature, Vhl-null mutation (i.e., UBCCreER/+Vhlfl/fl) had little effect on initial vessel branching, but it severely reduced arterial and venous branching at later stages. Interestingly, this mutation accelerated arterial maturation, as observed in retina vessel morphology and aberrant α-smooth muscle actin localization, particularly in vascular pericytes. RNA sequencing analysis identified gene expression changes within several key pathways, including Notch and smooth muscle cell contractility. Notch inhibition failed to reverse later-stage branching defects but rescued the accelerated arterialization. Retinal vessels harboring the type 2B Vhl mutation (i.e., UBCCreER/+Vhlfl/2B) displayed stage-specific changes in vessel branching and an advanced progression toward an arterial phenotype. Disrupting Notch signaling in type 2B mutants increased both artery and vein branching and restored arterial maturation toward nonmutant levels. By revealing differential effects of the null and type 2B Vhl mutations on vessel branching and maturation, these data may provide insight into the variability of VHL-associated vascular changes — particularly the heterogeneity and aggressiveness in ccRCC vessel growth — and also suggest Notch pathway targets for treating VHL syndrome.
Alexandra Arreola, Laura Beth Payne, Morgan H. Julian, Aguirre A. de Cubas, Anthony B. Daniels, Sarah Taylor, Huaning Zhao, Jordan Darden, Victoria L. Bautch, W. Kimryn Rathmell, John C. Chappell
Phosphatase and tensin homolog (PTEN) is an essential regulator of the differentiated vascular smooth muscle cell (SMC) phenotype. Our goal was to establish that PTEN loss promotes SMC dedifferentiation and pathological vascular remodeling in human atherosclerotic coronary arteries and nonatherosclerotic coronary arteries exposed to continuous-flow left ventricular assist devices (CF-LVADs). Arteries were categorized as nonatherosclerotic hyperplasia (NAH), atherosclerotic hyperplasia (AH), or complex plaque (CP). NAH coronary arteries from CF-LVAD patients were compared to NAH coronaries from non-LVAD patients. Intimal PTEN and SMC contractile protein expression was reduced compared with the media in arteries with NAH, AH, or CP. Compared with NAH, PTEN and SMC contractile protein expression was reduced in the media and intima of arteries with AH and CP. NAH arteries from CF-LVAD patients showed marked vascular remodeling and reduced PTEN and α-smooth muscle actin (αSMA) in medial SMCs compared with arteries from non-LVAD patients; this correlated with increased medial collagen deposition. Mechanistically, compared with ApoE–/– mice, SMC-specific PTEN-null/ApoE–/– double-knockout mice exhibited accelerated atherosclerosis progression and increased vascular fibrosis. By microarray and validated quantitative RT-PCR analysis, SMC PTEN deficiency promotes a global upregulation of proinflammatory and profibrotic genes. We propose that PTEN is an antiinflammatory, antifibrotic target that functions to maintain SMC differentiation. SMC loss of PTEN results in pathological vascular remodeling of human arteries.
Karen S. Moulton, Marcella Li, Keith Strand, Shawna Burgett, Penn McClatchey, Rebecca Tucker, Seth B. Furgeson, Sizhao Lu, Bruce Kirkpatrick, Joseph C. Cleveland, Raphael A. Nemenoff, Amrut V. Ambardekar, Mary C.M. Weiser-Evans
Several imaging modalities have been used to assess lymphatic function, including fluorescence microscopy, near-infrared fluorescence (NIRF) imaging, and Doppler optical coherence tomography (DOCT). They vary in how the mouse is positioned, the invasiveness of the experimental setup, and the volume of contrast agent injected. Here, we present how each of these experimental parameters affects functional measurements of collecting lymphatic vessels. First, fluorescence microscopy showed that supine mice have a statistically lower contraction frequency compared with mice sitting upright. To assess the effect of different injection volumes on these endpoints, mice were injected with 4, 10, or 20 μl of dye. The lowest frequencies were observed after 20-μl injections. Interestingly, lymph-flow DOCT revealed that although there was lower contraction frequency in mice injected with 20 μl versus 4 μl, mice showed a higher volumetric flow with a 20-μl injection. This indicates that contraction frequency alone is not sufficient to understand lymphatic transport. Finally, NIRF revealed that removing the skin reduced contraction frequency. Therefore, this study reveals how sensitive these techniques are to mouse position, removal of skin, and dye volume. Care should be taken when comparing results obtained under different experimental conditions.
Echoe M. Bouta, Cedric Blatter, Thomas A. Ruggieri, Eelco F.J. Meijer, Lance L. Munn, Benjamin J. Vakoc, Timothy P. Padera
Idiopathic pulmonary fibrosis (IPF) is a fatal disease of unknown etiology characterized by a compositionally and mechanically altered extracellular matrix. Poor understanding of the origin of α-smooth muscle actin (α-SMA) expressing myofibroblasts has hindered curative therapies. Though proposed as a source of myofibroblasts in mammalian tissues, identification of microvascular pericytes (PC) as contributors to α-SMA–expressing populations in human IPF and the mechanisms driving this accumulation remain unexplored. Here, we demonstrate enhanced detection of α-SMA+ cells coexpressing the PC marker neural/glial antigen 2 in the human IPF lung. Isolated human PC cultured on decellularized IPF lung matrices adopt expression of α-SMA, demonstrating that these cells undergo phenotypic transition in response to direct contact with the extracellular matrix (ECM) of the fibrotic human lung. Using potentially novel human lung–conjugated hydrogels with tunable mechanical properties, we decoupled PC responses to matrix composition and stiffness to show that α-SMA+ PC accumulate in a mechanosensitive manner independent of matrix composition. PC activated with TGF-β1 remodel the normal lung matrix, increasing tissue stiffness to facilitate the emergence of α-SMA+ PC via MKL-1/MTRFA mechanotranduction. Nintedanib, a tyrosine-kinase inhibitor approved for IPF treatment, restores the elastic modulus of fibrotic lung matrices to reverse the α-SMA+ phenotype. This work furthers our understanding of the role that microvascular PC play in the evolution of IPF, describes the creation of an ex vivo platform that advances the study of fibrosis, and presents a potentially novel mode of action for a commonly used antifibrotic therapy that has great relevance for human disease.
Parid Sava, Anand Ramanathan, Amelia Dobronyi, Xueyan Peng, Huanxing Sun, Adrian Ledesma-Mendoza, Erica L. Herzog, Anjelica L. Gonzalez
Infantile hemangioma (IH) is a vascular tumor that begins with rapid vascular proliferation shortly after birth, followed by vascular involution in early childhood. We have found that NOTCH3, a critical regulator of mural cell differentiation and maturation, is expressed in hemangioma stem cells (HemSCs), suggesting that NOTCH3 may function in HemSC-to–mural cell differentiation and pathological vessel stabilization. Here, we demonstrate that NOTCH3 is expressed in NG2+PDGFRβ+ perivascular HemSCs and CD31+GLUT1+ hemangioma endothelial cells (HemECs) in proliferating IHs and becomes mostly restricted to the αSMA+NG2loPDGFRβlo mural cells in involuting IHs. NOTCH3 knockdown in HemSCs inhibited in vitro mural cell differentiation and perturbed αSMA expression. In a mouse model of IH, NOTCH3 knockdown or systemic expression of the NOTCH3 inhibitor, NOTCH3 Decoy, significantly decreased IH blood flow, vessel caliber, and αSMA+ perivascular cell coverage. Thus, NOTCH3 is necessary for HemSC-to–mural cell differentiation, and adequate perivascular cell coverage of IH vessels is required for IH vessel stability.
Andrew K. Edwards, Kyle Glithero, Peter Grzesik, Alison A. Kitajewski, Naikhoba C.O. Munabi, Krista Hardy, Qian Kun Tan, Michael Schonning, Thaned Kangsamaksin, Jan K. Kitajewski, Carrie J. Shawber, June K. Wu
Fibrous cap smooth muscle cells (SMCs) protect atherosclerotic lesions from rupturing and causing thrombosis, while other plaque SMCs may have detrimental roles in plaque development. To gain insight into recruitment of different plaque SMCs, we mapped their clonal architecture in aggregation chimeras of eGFP+Apoe–/– and Apoe–/– mouse embryos and in mice with a mosaic expression of fluorescent proteins in medial SMCs that were rendered atherosclerotic by PCSK9-induced hypercholesterolemia. Fibrous caps in aggregation chimeras were found constructed from large, endothelial-aligned layers of either eGFP+ or nonfluorescent SMCs, indicating substantial clonal expansion of a few cells. Similarly, plaques in mice with SMC-restricted Confetti expression showed oligoclonal SMC populations with little intermixing between the progeny of different medial SMCs. Phenotypes comprised both ACTA2+ SMCs in the cap and heterogeneous ACTA2– SMCs in the plaque interior, including chondrocyte-like cells and cells with intracellular lipid and crystalline material. Fibrous cap SMCs were invariably arranged in endothelium-aligned clonal sheets, confirming results in the aggregation chimeras. Analysis of the clonal structure showed that a low number of local medial SMCs partake in atherosclerosis and that single medial SMCs can produce several different SMC phenotypes in plaque. The combined results show that few medial SMCs proliferate to form the entire phenotypically heterogeneous plaque SMC population in murine atherosclerosis.
Kevin Jacobsen, Marie Bek Lund, Jeong Shim, Stine Gunnersen, Ernst-Martin Füchtbauer, Mads Kjolby, Laura Carramolino, Jacob Fog Bentzon
Vascular calcification is a risk factor that predicts adverse cardiovascular complications of several diseases including atherosclerosis. Reduced dietary potassium intake has been linked to cardiovascular diseases such as hypertension and incidental stroke, although the underlying molecular mechanisms remain largely unknown. Using the ApoE-deficient mouse model, we demonstrated for the first time to our knowledge that reduced dietary potassium (0.3%) promoted atherosclerotic vascular calcification and increased aortic stiffness, compared with normal (0.7%) potassium–fed mice. In contrast, increased dietary potassium (2.1%) attenuated vascular calcification and aortic stiffness. Mechanistically, reduction in the potassium concentration to the lower limit of the physiological range increased intracellular calcium, which activated a cAMP response element–binding protein (CREB) signal that subsequently enhanced autophagy and promoted vascular smooth muscle cell (VSMC) calcification. Inhibition of calcium signals and knockdown of either CREB or ATG7, an autophagy regulator, attenuated VSMC calcification induced by low potassium. Consistently, elevated autophagy and CREB signaling were demonstrated in the calcified arteries from low potassium diet–fed mice as well as aortic arteries exposed to low potassium ex vivo. These studies established a potentially novel causative role of dietary potassium intake in regulating atherosclerotic vascular calcification and stiffness, and uncovered mechanisms that offer opportunities to develop therapeutic strategies to control vascular disease.
Yong Sun, Chang Hyun Byon, Youfeng Yang, Wayne E. Bradley, Louis J. Dell’Italia, Paul W. Sanders, Anupam Agarwal, Hui Wu, Yabing Chen
Blood pressure is regulated by extrinsic factors including noradrenaline, the sympathetic neurotransmitter that controls cardiovascular functions through adrenergic receptors. However, the fine-tuning system of noradrenaline signaling is relatively unknown. We here show that l-3,4-dihydroxyphenylalanine (L-DOPA), a precursor of catecholamines, sensitizes the vascular adrenergic receptor alpha1 (ADRA1) through activation of L-DOPA receptor GPR143. In WT mice, intravenous infusion of the ADRA1 agonist phenylephrine induced a transient elevation of blood pressure. This response was attenuated in Gpr143 gene–deficient (Gpr143–/y) mice. Specific knockout of Gpr143 in vascular smooth muscle cells (VSMCs) also showed a similar phenotype, indicating that L-DOPA directly modulates ADRA1 signaling in the VSMCs. L-DOPA at nanomolar concentrations alone produced no effect on the VSMCs, but it enhanced phenylephrine-induced vasoconstriction and intracellular Ca2+ responses. Phenylephrine also augmented the phosphorylation of extracellular signal–regulated kinases in cultured VSMCs from WT but not Gpr143–/y mice. In WT mice, blood pressure increased during the transition from light-rest to dark-active phases. This elevation was not observed in Gpr143–/y mice. Taken together, our findings provide evidence for L-DOPA/GPR143 signaling that exerts precursor control of sympathetic neurotransmission through sensitizing vascular ADRA1.
Daiki Masukawa, Motokazu Koga, Anna Sezaki, Yuka Nakao, Yuji Kamikubo, Tatsuo Hashimoto, Yuki Okuyama-Oki, Aderemi Caleb Aladeokin, Fumio Nakamura, Utako Yokoyama, Hiromichi Wakui, Hiroshi Ichinose, Takashi Sakurai, Satoshi Umemura, Koichi Tamura, Yoshihiro Ishikawa, Yoshio Goshima
Clinical trials in patients with macular edema due to diabetic retinopathy or retinal vein occlusion (RVO) have shown that suppression of VEGF not only improves macular edema, but also reopens closed retinal vessels, prevents progression of vessel closure, and improves retinopathy. In this study, we show the molecular basis for those clinical observations. Increased retinal levels of VEGF in mice cause plugging of retinal vessels with leukocytes, vessel closure, and hypoxia. Suppression of VEGF reduces leukocyte plugging, causing reperfusion of closed vessels. Activation of VEGFR1 contributes to leukocyte recruitment, because it is significantly reduced by an anti-VEGFR1–neutralizing antibody. High VEGF increases transcriptional activity of NF-κB and expression of NF-κB target genes, particularly Vcam1. Injection of an anti-VCAM-1–neutralizing antibody reduces VEGF-induced leukocyte plugging. These data explain the broad range of benefits obtained by VEGF suppression in patients with ischemic retinopathies, provide an important insight into the pathogenesis of RVO and diabetic retinopathy, and suggest that sustained suppression of VEGF early in the course of these diseases may prevent vessel closure, worsening ischemia, and disease progression. This study also identifies VEGFR1 and VCAM-1 as molecular targets whose suppression could supplement VEGF neutralization for treatment of RVO and diabetic retinopathy.
Yuanyuan Liu, Jikui Shen, Seth D. Fortmann, Jiangxia Wang, Dietmar Vestweber, Peter A. Campochiaro
No posts were found with this tag.