Abstract
Marfan syndrome is a connective tissue disorder caused by mutations in the gene encoding fibrillin-1 (FBN1). A dominant-negative mechanism has been inferred based upon dominant inheritance, mulitimerization of monomers to form microfibrils, and the dramatic paucity of matrix-incorporated fibrillin-1 seen in heterozygous patient samples. Yeast artificial chromosome–based transgenesis was used to overexpress a disease-associated mutant form of human fibrillin-1 (C1663R) on a normal mouse background. Remarkably, these mice failed to show any abnormalities of cellular or clinical phenotype despite regulated overexpression of mutant protein in relevant tissues and developmental stages and direct evidence that mouse and human fibrillin-1 interact with high efficiency. Immunostaining with a human-specific mAb provides what we believe to be the first demonstration that mutant fibrillin-1 can participate in productive microfibrillar assembly. Informatively, use of homologous recombination to generate mice heterozygous for a comparable missense mutation (C1039G) revealed impaired microfibrillar deposition, skeletal deformity, and progressive deterioration of aortic wall architecture, comparable to characteristics of the human condition. These data are consistent with a model that invokes haploinsufficiency for WT fibrillin-1, rather than production of mutant protein, as the primary determinant of failed microfibrillar assembly. In keeping with this model, introduction of a WT FBN1 transgene on a heterozygous C1039G background rescues aortic phenotype.
Authors
Daniel P. Judge, Nancy J. Biery, Douglas R. Keene, Jessica Geubtner, Loretha Myers, David L. Huso, Lynn Y. Sakai, Harry C. Dietz
Abstract
Peroxisome proliferator–activated receptor γ (PPARγ), the molecular target of a class of insulin sensitizers, regulates adipocyte differentiation and lipid metabolism. A dominant negative P467L mutation in the ligand-binding domain of PPARγ in humans is associated with severe insulin resistance and hypertension. Homozygous mice with the equivalent P465L mutation die in utero. Heterozygous mice grow normally and have normal total adipose tissue weight. However, they have reduced interscapular brown adipose tissue and intra-abdominal fat mass, and increased extra-abdominal subcutaneous fat, compared with wild-type mice. They have normal plasma glucose levels and insulin sensitivity, and increased glucose tolerance. However, during high-fat feeding, their plasma insulin levels are mildly elevated in association with a significant increase in pancreatic islet mass. They are hypertensive, and expression of the angiotensinogen gene is increased in their subcutaneous adipose tissues. The effects of P465L on blood pressure, fat distribution, and insulin sensitivity are the same in both male and female mice regardless of diet and age. Thus the P465L mutation alone is sufficient to cause abnormal fat distribution and hypertension but not insulin resistance in mice. These results provide genetic evidence for a critical role for PPARγ in blood pressure regulation that is not dependent on altered insulin sensitivity.
Authors
Yau-Sheng Tsai, Hyo-Jeong Kim, Nobuyuki Takahashi, Hyung-Suk Kim, John R. Hagaman, Jason K. Kim, Nobuyo Maeda
Abstract
Abdominal aortic aneurysms (AAAs) cause death due to complications related to expansion and rupture. The underlying mechanisms that drive AAA development remain largely unknown. We recently described evidence for a shift toward T helper type 2 (Th2) cell responses in human AAAs compared with stenotic atheromas. To evaluate putative pathways in AAA formation, we induced Th1- or Th2-predominant cytokine environments in an inflammatory aortic lesion using murine aortic transplantation into WT hosts or those lacking the receptors for the hallmark Th1 cytokine IFN-γ, respectively. Allografts in WT recipients developed intimal hyperplasia, whereas allografts in IFN-γ receptor–deficient (GRKO) hosts developed severe AAA formation associated with markedly increased levels of MMP-9 and MMP-12. Allografts in GRKO recipients treated with anti–IL-4 antibody to block the characteristic IL-4 Th2 cytokine or allografts in GRKO hosts also congenitally deficient in IL-4 did not develop AAA and likewise exhibited attenuated collagenolytic and elastolytic activities. These observations demonstrate an important dichotomy between cellular immune responses that induce IFN-γ– or IL-4–dominated cytokine environments. The findings establish important regulatory roles for a Th1/Th2 cytokine balance in modulating matrix remodeling and have important implications for the pathophysiology of AAAs and arteriosclerosis.
Authors
Koichi Shimizu, Masayoshi Shichiri, Peter Libby, Richard T. Lee, Richard N. Mitchell
Abstract
Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The αvβ3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111–labeled (111In-labeled) αvβ3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted αvβ3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled αvβ3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission–computed tomographic (SPECT) imaging with the 111In-labeled αvβ3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the αvβ3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an 111In-labeled αvβ3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.
Authors
David F. Meoli, Mehran M. Sadeghi, Svetlana Krassilnikova, Brian N. Bourke, Frank J. Giordano, Donald P. Dione, Haili Su, D. Scott Edwards, Shuang Liu, Thomas D. Harris, Joseph A. Madri, Barry L. Zaret, Albert J. Sinusas
Abstract
Although arterial bifurcations are frequent sites for obstructive atherosclerotic lesions, the optimal approach to these lesions remains unresolved. Benchtop models of arterial bifurcations were analyzed for flow disturbances known to correlate with vascular disease. These models possess an adaptable geometry capable of simulating the course of arterial disease and the effects of arterial interventions. Chronic in vivo studies evaluated the effect of flow disturbances on the pattern of neointimal hyperplasia. Acute in vivo studies helped propose a mechanism that bridges the early mechanical stimulus and the late tissue effect. Side-branch (SB) dilation adversely affected flow patterns in the main branch (MB) and, as a result, the long-term MB patency of stents implanted in pig arteries. Critical to this effect is chronic MB remodeling that seems to compensate for an occluded SB. Acute leukocyte recruitment was directly influenced by the changes in flow patterns, suggesting a link between flow disturbance on the one hand and leukocyte recruitment and intimal hyperplasia on the other. It is often impossible to simultaneously maximize the total cross-sectional area of both branches and to minimize flow disturbance in the MB. The apparent trade-off between these two clinically desirable goals may explain many of the common failure modes of bifurcation stenting.
Authors
Yoram Richter, Adam Groothuis, Philip Seifert, Elazer R. Edelman
Abstract
Recent evidence indicates that vascular progenitor cells may be the source of smooth muscle cells (SMCs) that accumulate in atherosclerotic lesions, but the origin of these progenitor cells is unknown. To explore the possibility of vascular progenitor cells existing in adults, a variety of tissues from ApoE-deficient mice were extensively examined. Immunohistochemical staining revealed that the adventitia in aortic roots harbored large numbers of cells having stem cell markers, e.g., Sca-1+ (21%), c-kit+ (9%), CD34+ (15%), and Flk1+ cells (4%), but not SSEA-1+ embryonic stem cells. Explanted cultures of adventitial tissues using stem cell medium displayed a heterogeneous outgrowth, for example, islands of round-shaped cells surrounded by fibroblast-like cell monolayers. Isolated Sca-1+ cells were able to differentiate into SMCs in response to PDGF-BB stimulation in vitro. When Sca-1+ cells carrying the LacZ gene were transferred to the adventitial side of vein grafts in ApoE-deficient mice, β-gal+ cells were found in atherosclerotic lesions of the intima, and these cells enhanced the development of the lesions. Thus, a large population of vascular progenitor cells existing in the adventitia can differentiate into SMCs that contribute to atherosclerosis. Our findings indicate that ex vivo expansion of these progenitor cells may have implications for cellular, genetic, and tissue engineering approaches to vascular disease.
Authors
Yanhua Hu, Zhongyi Zhang, Evelyn Torsney, Ali R. Afzal, Fergus Davison, Bernhard Metzler, Qingbo Xu
Abstract
Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40–/connexin45+ cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system.
Authors
Patrick Y. Jay, Brett S. Harris, Colin T. Maguire, Antje Buerger, Hiroko Wakimoto, Makoto Tanaka, Sabina Kupershmidt, Dan M. Roden, Thomas M. Schultheiss, Terrence X. O’Brien, Robert G. Gourdie, Charles I. Berul, Seigo Izumo
Abstract
Hypertension is the most prevalent risk factor for cardiovascular diseases, present in almost 30% of adults. A key element in the control of vascular tone is the large-conductance, Ca2+-dependent K+ (BK) channel. The BK channel in vascular smooth muscle is formed by an ion-conducting α subunit and a regulatory β1 subunit, which couples local increases in intracellular Ca2+ to augmented channel activity and vascular relaxation. Our large population-based genetic epidemiological study has identified a new single-nucleotide substitution (G352A) in the β1 gene (KCNMB1), corresponding to an E65K mutation in the protein. This mutation results in a gain of function of the channel and is associated with low prevalence of moderate and severe diastolic hypertension. BK-β1E65K channels showed increased Ca2+ sensitivity, compared with wild-type channels, without changes in channel kinetics. In conclusion, the BK-β1E65K channel might offer a more efficient negative-feedback effect on vascular smooth muscle contractility, consistent with a protective effect of the K allele against the severity of diastolic hypertension.
Authors
José M. Fernández-Fernández, Marta Tomás, Esther Vázquez, Patricio Orio, Ramón Latorre, Mariano Sentí, Jaume Marrugat, Miguel A. Valverde
Abstract
The cardiac sympathetic nerve plays an important role in regulating cardiac function, and nerve growth factor (NGF) contributes to its development and maintenance. However, little is known about the molecular mechanisms that regulate NGF expression and sympathetic innervation of the heart. In an effort to identify regulators of NGF in cardiomyocytes, we found that endothelin-1 specifically upregulated NGF expression in primary cultured cardiomyocytes. Endothelin-1–induced NGF augmentation was mediated by the endothelin-A receptor, Giβγ, PKC, the Src family, EGFR, extracellular signal–regulated kinase, p38MAPK, activator protein-1, and the CCAAT/enhancer-binding protein δ element. Either conditioned medium or coculture with endothelin-1–stimulated cardiomyocytes caused NGF-mediated PC12 cell differentiation. NGF expression, cardiac sympathetic innervation, and norepinephrine concentration were specifically reduced in endothelin-1–deficient mouse hearts, but not in angiotensinogen-deficient mice. In endothelin-1–deficient mice the sympathetic stellate ganglia exhibited excess apoptosis and displayed loss of neurons at the late embryonic stage. Furthermore, cardiac-specific overexpression of NGF in endothelin-1–deficient mice overcame the reduced sympathetic innervation and loss of stellate ganglia neurons. These findings indicate that endothelin-1 regulates NGF expression in cardiomyocytes and plays a critical role in sympathetic innervation of the heart.
Authors
Masaki Ieda, Keiichi Fukuda, Yasuyo Hisaka, Kensuke Kimura, Haruko Kawaguchi, Jun Fujita, Kouji Shimoda, Eiko Takeshita, Hideyuki Okano, Yukiko Kurihara, Hiroki Kurihara, Junji Ishida, Akiyoshi Fukamizu, Howard J. Federoff, Satoshi Ogawa
Abstract
Ischemia resulting from myocardial infarction (MI) promotes VEGF expression, leading to vascular permeability (VP) and edema, a process that we show here contributes to tissue injury throughout the ventricle. This permeability/edema can be assessed noninvasively by MRI and can be observed at the ultrastructural level as gaps between adjacent endothelial cells. Many of these gaps contain activated platelets adhering to exposed basement membrane, reducing vessel patency. Following MI, genetic or pharmacological blockade of Src preserves endothelial cell barrier function, suppressing VP and infarct volume, providing long-term improvement in cardiac function, fibrosis, and survival. To our surprise, an intravascular injection of VEGF into healthy animals, but not those deficient in Src, induced similar endothelial gaps, VP, platelet plugs, and some myocyte damage. Mechanistically, we show that quiescent blood vessels contain a complex involving Flk, VE-cadherin, and β-catenin that is transiently disrupted by VEGF injection. Blockade of Src prevents disassociation of this complex with the same kinetics with which it prevents VEGF-mediated VP/edema. These findings define a molecular mechanism to account for the Src requirement in VEGF-mediated permeability and provide a basis for Src inhibition as a therapeutic option for patients with acute MI.
Authors
Sara Weis, Satoshi Shintani, Alberto Weber, Rudolf Kirchmair, Malcolm Wood, Adrianna Cravens, Heather McSharry, Atsushi Iwakura, Young-sup Yoon, Nathan Himes, Deborah Burstein, John Doukas, Richard Soll, Douglas Losordo, David Cheresh
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ISSN: 0021-9738 (print), 1558-8238 (online)