Neural architecture search via progressive partial connection with attention mechanism.

Differentiable architecture search requires a larger computational consumption during architecture search, and there exists the depth gap problem under deeper network architecture. In this paper, we propose an attention-based progressive partially connected neural architecture search method (PPCAtt-NAS) to address these two issues. First, we introduce a progressive search strategy in the architecture search phase, build up the sophistication of the architecture gradually and perform path-level pruning in stages to bridge the depth gap. Second, we adopt a partial search scheme that performs channel-level partial sampling of the network architecture to further reduce the computational complexity of the architecture search. In addition, an attention mechanism is devised to improve the architecture search capability by enhancing the relevance between the feature channels. Finally, we conduct extensive comparison experiments with state-of-the-art methods on several public datasets, and our method is able to present higher architecture performance.

TRIM29 promotes the progression of colorectal cancer by suppressing EZH2 degradation.

Colorectal cancer (CRC) is commonly diagnosed at the advanced stage and has a high mortality rate. Tripartite Motif Containing 29 (TRIM29) is an oncogene in numerous malignancies including CRC. However, the molecular mechanism of TRIM29 is largely unknown. In this study, we investigated the biological functions of TRIM29 and the underlying mechanisms. The expression of TRIM29 and Enhancer of Zeste Homolog 2 (EZH2) was predicted using the bioinformatic analysis and measured using a quantitative real-time polymerase chain reaction (PCR) and immunohistochemical assay. The biological functions of TRIM29 were analyzed using a cell counting kit-8, EdU and transwell assays, scratch test, and flow cytometry. The interaction between TRIM29 and EZH2 was assessed using protein immunoprecipitation. The stability of EZH2 was evaluated by treating it with cycloheximide. Our results indicated that TRIM29 levels were upregulated in CRC. Overexpression of TRIM29 promoted CRC cell proliferation and migration and suppressed apoptosis. The opposite result was obtained when TRIM29 was silenced. TRIM29 interacted with EZH2 mechanically and enhanced the protein stability of EZH2. Depletion of EZH2 reversed the effects of TRIM29, regarding its biological behaviors. Moreover, downregulation of TRIM29 inhibited tumor growth and improved the histopathological prognosis. In conclusion, EZH2 interacted with silenced TRIM29 to suppress its stability, thereby inhibiting cell proliferation, migration, and tumor growth, and promoting apoptosis in CRC. Our findings suggested that TRIM29 is a promising target for CRC therapy.

Neural architecture search based on dual attention mechanism for image classification.

Deep learning neural networks based on the manual design for image classification tasks usually require a large amount of a priori knowledge and experience from experts; thus, research on designing neural network architectures automatically has been widely performed. The neural architecture search (NAS) method based on the differentiable architecture search (DARTS) ignores the interrelationships within the searched network architecture cells. The optional operations in the architecture search space lack diversity, and the large parametric and non-parametric operations in the search space make the search process inefficient. We propose a NAS method based on a dual attention mechanism (DAM-DARTS). An improved attention mechanism module is introduced to the cell of the network architecture to deepen the interrelationships between the important layers within the architecture by enhancing the attention between them, which improves the accuracy of the architecture and reduces the architecture search time. We also propose a more efficient architecture search space by adding attention operations to increase the complex diversity of the searched network architectures and reduce the computational cost consumed in the search process by reducing non-parametric operations. Based on this, we further analyze the impact of changing some operations in the architecture search space on the accuracy of the architectures. Through extensive experiments on several open datasets, we demonstrate the effectiveness of the proposed search strategy, which is highly competitive with other existing neural network architecture search methods.

Regulation of endothelial nitric oxide synthase in cardiac remodeling.

OBJECTIVES: Our previous study demonstrated that endothelial nitric oxide synthase (eNOS) gene serves as a candidate for modifiers of hypertrophic cardiomyopathy (HCM), which alters severity of HCM phenotypes. Herein, we sought to further elucidate the role of eNOS on cardiac myocyte hypertrophy and fibrosis, the major phenotypes of HCM. METHODS: Male eNOS-deficient mice (eNOS-/-) and wild type control mice (eNOS+/+, C57B1/6 J) were used in this study. Myocyte size was analyzed in hematoxylin/eosin stained sections using an image analyzing system. Cardiac ß-myosin heavy chain (ß-MHC) and α-skeletal actin (α-SKA) levels, markers of myocyte hypertrophy were evaluated by Western blot. Cardiac collagen volume fraction (CVF) was examined in picrosirius red stained section using an image analyzing system. Cardiac expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-ß1), markers of fibrosis, were determined by Western blot. RESULTS: Compared to eNOS+/+ mice, we found that; 1) myocyte size was significantly increased in eNOS-/- mice; 2) cardiac expression of ß-MHC was markedly elevated, while α-SKA levels remained unchanged in eNOS-/- mice; 3) cardiac total and interstitial CVF levels were significantly higher in eNOS-/- mice; and 4) cardiac TIMP-1 levels were significantly greater in eNOS-/- mice, however, cardiac TGF-ß1 was not differently expressed between the two groups. CONCLUSION: The current study revealed that eNOS plays a beneficial role in cardiac remodeling, preventing the heart from development of myocyte hypertrophy and cardiac fibrosis. These findings support our previous report that eNOS may modify the severity of HCM phenotypes.

Characterizing modifier genes of cardiac fibrosis phenotype in hypertrophic cardiomyopathy.

BACKGROUND: Clinical phenotypes of hypertrophic cardiomyopathy (HCM) vary greatly even among patients with the same gene mutations. This variability is largely regulated by unidentified modifier loci. The purpose of the study is to identify modifier genes for cardiac fibrosis-a major phenotype of HCM-using the BXD family, a murine cohort. METHODS: The relative severity of cardiac fibrosis was estimated by quantitation of cardiac collagen volume fraction (CCVF) across 66 members of the BXD family. Quantitative trait locus (QTL) mapping for cardiac fibrosis was done using GeneNetwork. Candidate modifier loci and genes associated with fibrosis were prioritized based on an explicit scoring system. Networks of correlation between fibrosis and cardiac transcriptomes were evaluated to generate causal models of disease susceptibility. RESULTS: CCVF levels varied greatly within this family. Interval mapping identified a significant CCVF-related QTL on chromosome (Chr) 2 in males, and a significant QTL on Chr 4 Mb in females. The scoring system highlighted two strong candidate genes in the Chr 2 locus-Nek6 and Nr6a1. Both genes are highly expressed in the heart. Cardiac Nek6 mRNA levels are significantly correlated with CCVF. Nipsnap3b and Fktn are lead candidate genes for the Chr 4 locus, and both are also highly expressed in heart. Cardiac Nipsnap3b gene expression correlates well with CCVF. CONCLUSION: Our study demonstrated that candidate modifier genes of cardiac fibrosis phenotype in HCM are different in males and females. Nek6 and Nr6a1 are strong candidates in males, while Nipsnap3b and Fktn are top candidates in females.

Identifying modifier genes for hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) severity greatly varies among patients even with the same HCM gene mutations. This variation is largely regulated by modifier gene(s), which, however, remain largely unknown. The current study is aimed to identify modifier genes using BXD strains, a large murine genetic reference population (GRP) derived from crosses between C57BL/6 J (B6) and D2 DBA/2 J (D2) mice. D2 mice natualy carrythe genetic basis and phenotypes of HCM. METHODS: Myocardial hypertrophy, the major phenotype of HCM, was determined by cardiomyocyte size on cardiac sections in 30 BXD strains, and their parental B6 and D2 strains and morphometric analysis was performed. Quantitative Trait Locus (QTL) mapping for cardiomyocyte sizes was conducted with WebQTL in GeneNetwork. Correlation of cardiomyocyte size and cardiac gene expression in BXDs accessed from GeneNetwork were evaluated. QTL candidate genes associated with cardiomyocyte sizes were prioritized based on the score system. RESULTS: Cardiomyocyte size varied significantly among BXD strains. Interval mapping on cardiomyocyte size data showed a significant QTL on chromosome (Chr) 2 at 66- 73.5 Mb and a suggestive QTL on Chr 5 at 20.9-39.7 Mb. Further score system revealed a high QTL score for Xirp2 in Chr 2. Xirp2 encodes xin actin-binding repeat containing 2, which is highly expressed in cardiac tissue and associate with cardiomyopathy and heart failure. In Chr5 QTL, Nos3, encoding nitric oxide synthase 3, received the highest score, which is significantly correlated with cardiomyocyte size. CONCLUSION: These results indicate that Xirp2 and Nos3 serve as novel candidate modifier genes for myocardial hypertrophy in HCM. These candidate genes will be validated in our future studies.

Molecular and Cellular Effect of Angiotensin 1-7 on Hypertensive Kidney Disease.

BACKGROUND: Studies implicate that angiotensin 1-7 (Ang1-7) imparts protective effects in the kidney. However, its relevance in hypertensive kidney disease is not fully understood. The purpose of this study was to explore the role of Ang1-7 on renal damage/remodeling during hypertension and its potential underlying molecular-cellular mechanisms. METHODS: Hypertension was induced in adult Sprague-Dawley rats by infusion of aldosterone (ALDO; 0.75 µg/hour) for 4 weeks with or without co-treatment of Ang1-7 (1 mg/kg/day). Untreated rats served as controls. Systolic blood pressure was monitored by tail-cuff technique. Renal fibrosis was evaluated by picrosirius red staining and renal collagen volume fraction was quantitated using imaging analyzing system. The expression of profibrotic factors [transforming growth factor-ß1 (TGF-ß1), platelet-derived growth factor-D (PDGF-D), fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor-D (VEGF-D), and tissue inhibitors of metalloproteinases (TIMPs)] and free radical producing enzymes (inducible nitric oxide synthase and nicotinamide adenine dinucleotide phosphate [NADPH] oxidase) in the kidney were examined by reverse transcription-polymerase chain reaction and western blot. Renal oxidative stress was assessed by malondialdehyde (MDA) measurement. RESULTS: Chronic ALDO infusion caused hypertension and hypertensive renal disease represented as glomerular damage/sclerosis. Ang1-7 co-treatment did not affect blood pressure in ALDO-treated rats, but significantly attenuated the glomerular damage/fibrosis. ALDO treatment significantly elevated renal expression of profibrogenic factors, including TGF-ß1, TIMP-1/TIMP-2, FGF-1, PDGF-D, and VEGF-D, whereas Ang1-7 co-treatment significantly reduced renal TGF-ß1, TIMP-1/TIMP-2, and FGF-1, but not PDGF-D and VEGF-D. Furthermore, ALDO infusion elevated NADPH oxidase (gp91phox) and MDA in the kidney, which was attenuated by Ang1-7 co-treatment. CONCLUSIONS: Ang1-7 plays a protective role in the hypertensive kidney disease independent of blood pressure. The beneficial effects of Ang1-7 are likely mediated via suppressing TGF-ß/FGF-1 pathways and oxidative stress.

Cardiovascular Interactions between Fibroblast Growth Factor-23 and Angiotensin II.

Both the activation of the renin angiotensin aldosterone system (RAAS) and elevations of circulating Fibroblast Growth Factor-23 (FGF-23) have been implicated in the pathogenesis of left ventricular hypertrophy (LVH) in chronic kidney disease. To investigate potential cross-talk between RAAS and FGF-23, we administered angiotensin II (Ang II) to wild-type rodents and the Hyp mouse model of excess FGF-23. Ang II administration for four weeks to wild-type rodents resulted in significant increases in systolic blood pressure and LVH. Unexpectedly, FGF-23 circulating levels were increased by 1.5-1.7 fold in Ang II treated animals. In addition, Ang II treatment increased expression of FGF-23 message levels in bone, the predominant tissue for FGF-23 production, and induced expression of FGF-23 and its co-receptor α-Klotho in the heart, which normally does not express FGF-23 or α-Klotho in physiologically relevant levels. Hyp mice with elevated FGF-23 exhibited increased blood pressure and LVH at baseline. Ang II administration to Hyp mice resulted further increments in blood pressure and left ventricular hypertrophy, consistent with additive cardiovascular effects. These findings suggest that FGF-23 may participate in unexpected systemic and paracrine networks regulating hemodynamic and myocardial responses.

Differential Expression of Hypertensive Phenotypes in BXD Mouse Strains in Response to Angiotensin II.

BACKGROUND: Besides environmental risk factors, genetic factors play a crucial role in the pathogenesis of primary hypertension. The current study is to unravel whether hypertensive phenotypes vary in mice with different genetic background. METHODS: Hypertension was induced in C57BL/6J (B6), DBA/2J (D2), and 25 BXD strains by administrating angiotensin (Ang)II (2.5 mg/kg/day infused by osmotic minipump) for 4 weeks. Systolic blood pressure was monitored before (baseline) and after 4 weeks of AngII treatment by tail cuff. Cardiac and renal fibrosis was evaluated by picrosirius red staining and collagen volume fraction (CVF) was quantitated using imaging analyzing system; cardiac transforming growth factor (TGF)-ß gene expression was monitored by RT-PCR, and inflammatory response was detected by immunohistochemical ED-1 staining. RESULTS: AngII infusion caused hypertension in all strains. However, blood pressure elevation was more evident in the D2 strain than the B6 group, while it was widely variable among BXD strains. Furthermore, chronic AngII treatment lead to development of hypertensive cardiac and renal diseases. Cardiac and renal CVF levels in the D2 strain was significantly higher than the B6 cohort, whereas these varied vastly across BXD strains. Moreover, cardiac TGF-ß mRNA levels were markedly diverse among various mouse strains. CONCLUSION: Our study unequivocally demonstrates that in response to AngII, BXDs with different genetic background expressed hypertension phenotypes with varied degree in severity. It implicates that genomics contribute to pathogenesis of primary hypertension. Building upon the genotype and hypertensive phenotypes, the BXD cohort can be further exploited experimentally to identify genes that influence blood pressure.

Differential Regulatory Role of Soluble Klothos on Cardiac Fibrogenesis in Hypertension.

BACKGROUND: Soluble Klotho functions as an endocrine factor that plays important roles in a variety of pathophysiological processes. Soluble Klotho contains 130 KDa and 65 KDa isoforms. However, their distinct individual functional heterogeneity remains uncertain. Herein, we investigated the regulatory role of two soluble Klothos on cardiac fibrogenic responses. METHODS AND RESULTS: The effect of soluble Klothos on myofibroblast differentiation, proliferation, and collagen synthesis/degradation were examined in cultured mouse cardiac myofibroblasts. The role of 130 KDa Klotho on fibrosis in hypertensive heart disease were examined in wild type (WT) and Klotho transgenic (Tg/+) mice receiving chronic angiotensin (Ang)II infusion. Our in vitro studies revealed that addition of 130 KDa soluble Klotho isoform increased collagen synthesis in a dose dependent manner. Furthermore, 130 KDa Klotho significantly stimulated myofibroblast differentiation, proliferation, and ERK phosphorylation, which were abolished by fibroblast growth factor (FGF) receptor antagonist (SU5402). In contrast, 65 KDa soluble Klotho treatment significantly suppressed myofibroblast proliferation and collagen synthesis. In vivo study further demonstrated that chronic AngII infusion lead to cardiac fibrosis in both WT and Tg/+ mice. However, cardiac collagen, TGF-ß1, TIMP-2, and α-smooth muscle actin (SMA) levels were markedly upregulated in Tg/+ mice compared to WT cohort. CONCLUSION: Taken together, these findings implicate that 130 KDa soluble Klotho plays a stimulatory role in cardiac myofibroblast growth and activity through FGF pathway, whereas 65 KDa soluble Klotho exerts an anti-fibrotic effect in cardiac myofibroblasts. Thus, two distinct isoforms of soluble Klotho appear to play the counter-regulatory roles in cardiac fibrogenic responses.

Vascular endothelial growth factor-D mediates fibrogenic response in myofibroblasts.

Vascular endothelial growth factor (VEGF)-D is a crucial mediator of angiogenesis. Following myocardial infarction (MI), cardiac VEGF-D and VEGF receptor (VEGFR)-3 are significantly upregulated. In addition to endothelial cells, myofibroblasts at the site of MI highly express VEGFR-3, implicating the involvement of VEGF-D in cardiac fibrogenesis that promotes repair and remodeling. The aim of the current study was to further explore the critical role of VEGF-D in fibrogenic response in myofibroblasts. Myofibroblast proliferation, migration, collagen synthesis, and degradation were investigated in cultured cardiac myofibroblasts subjected to VEGF-D with/without VEGFR antagonist or ERK inhibitor. Vehicle-treated cells served as controls. Myofibroblast proliferation and migration were detected by BrdU assay and Boyden Chamber method, respectively. Expression of type I collagen, metalloproteinase (MMP)-2/-9, tissue inhibitor of MMP (TIMP)-1/-2, and ERK phosphorylation were evaluated by Western blot analyses. Our results revealed that compared to controls, (1) VEGF-D significantly increased myofibroblast proliferation and migration; (2) VEGF-D significantly upregulated type I collagen synthesis in a dose- and time-dependent manner; (3) VEGFR antagonist abolished VEGF-D-induced myofibroblast proliferation and type I collagen release; (4) VEGF-D stimulated MMP-2/-9 and TIMP-1/-2 synthesis; (5) VEGF-D activated ERK phosphorylation; and (6) ERK inhibitor abolished VEGF-D-induced myofibroblast proliferation and type I collagen synthesis. Our in vitro studies have demonstrated that VEGF-D serves as a crucial profibrogenic mediator by stimulating myofibroblast growth, migration and collagen synthesis. Further studies are underway to determine the role of VEGF-D in fibrous tissue formation during cardiac repair following MI.

A Murine Hypertrophic Cardiomyopathy Model: The DBA/2J Strain.

Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including ß-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens.

VEGF-C/VEGFR-3 pathway promotes myocyte hypertrophy and survival in the infarcted myocardium.

BACKGROUND: Numerous studies have shown that in addition to angio/lymphangiogenesis, the VEGF family is involved in other cellular actions. We have recently reported that enhanced VEGF-C and VEGFR-3 in the infarcted rat myocardium, suggesting the paracrine/autocrine function of VEGF-C on cardiac remodeling. The current study was designed to test the hypothesis that VEGF-C regulates cardiomyocyte growth and survival in the infarcted myocardium. METHODS AND RESULTS: Gene profiling and VEGFR-3 expression of cardiomyocytes were assessed by laser capture microdissection/microarray and immunohistochemistry in the normal and infarcted myocardium. The effect of VEGF-C on myocyte hypertrophy and apoptosis during normoxia and hypoxia was detected by RT-PCR and western blotting in cultured rat neonatal cardiomyocytes. VEGFR-3 was minimally expressed in cardiomyocytes of the normal and noninfarcted myocardium, while markedly elevated in the surviving cardiomyocytes of the infarcted myocardium and border zone. Genes altered in the surviving cardiomyocytes were associated with the networks regulating cellular growth and survival. VEGF-C significantly increased the expression of atrial natriuretic factor (ANP), brain natriuretic factor (BNP), and ß-myosin heavy chain (MHC), markers of hypertrophy, in neonatal cardiomyocytes. Hypoxia caused neonatal cardiomyocyte atrophy, which was prevented by VEGF-C treatment. Hypoxia significantly enhanced apoptotic mediators, including cleaved caspase 3, 8, and 9, and Bax in neonatal cardiomyocytes, which were abolished by VEGF-C treatment. CONCLUSION: Our findings indicate that VEGF-C/VEGFR-3 pathway exerts a beneficial role in the infarcted myocardium by promoting compensatory cardiomyocyte hypertrophy and survival.

Angiotensin 1-7 promotes cardiac angiogenesis following infarction.

Angiogenesis is central to cardiac repair following myocardial infarction (MI). Cardiac angiotensin converting enzyme (ACE)2 significantly increased postMI, which is coincident with activated angiogenesis. The function of ACE2 is to generate angiotensin (Ang)1-7, an active peptide with cellular actions mediated by Mas receptors. The current study is to determine whether Ang(1-7) is involved in cardiac angiogenesis and facilitates cardiac repair. In the first portion of the study, the temporal expressions of cardiac ACE2 and Mas receptors were detected in rats with MI. In the second portion, MI rats were treated with or without a Mas receptor antagonist, A779 (1mg/kg/day given by minipump) for 7 days. Vascular density and expression of angiogenic mediators in the infarcted myocardium and cardiac function were examined. Compared to controls, ACE2 and Mas receptor levels were significantly increased in the infarcted myocardium for 4 weeks of the observation period. Newly formed vessels were evident in the infarcted myocardium at day 7. Mas receptor blockade significantly reduced vascular density in the infarcted myocardium and impaired ventricular function. In addition, A779 treatment significantly suppressed the cardiac expressions of vascular endothelial growth factor (VEGF)-D and matrix metalloproteinase (MMP)-9 but not expression of other angiogenic mediators, including monocyte Chemoattractant protein (MCP-1), VEGF-C, transforming growth factor (TGF)-ß1 and integrin ß3. These observations indicate that Ang(1-7) promotes angiogenesis via stimulating the expression of cardiac VEGF-D and MMP-9, thus facilitating cardiac repair and ventricular function.

Platelet-derived growth factor blockade on cardiac remodeling following infarction.

Cardiac repair and remodeling occur following myocardial infarction (MI). Our previous study demonstrated that platelet-derived growth factor (PDGF)-A/-D and PDGF receptors (PDGFR) are increased in the infarcted heart, with cells expressing PDGFR primarily endothelial and fibroblast-like cells. In the present study, we tested the hypothesis that PDGF contributes to cardiac angiogenesis and fibrogenesis post-MI. Rats with experimental MI were treated with either a PDGFR antagonist (Imatinib, 40 mg/kg/day) or vehicle by gavage, and sham-operated rats served as the controls. Cardiac fibrogenesis, angiogenesis, and ventricular function were detected at weeks 1 and 4 post-MI. We found that (1) transforming growth factor (TGF)-ß1, tissue inhibitors of metalloproteinases (TIMP)-1/-2, and type I collagen mRNA were all significantly increased in the infarcted heart at week 1 post-MI, while PDGFR blockade significantly reduced these fibrogenic mediators in the noninfarcted myocardium as compared to controls; (2) fibrosis developed in both the infarcted and noninfarcted myocardium at week 4 with PDGFR blockade significantly suppressing collagen volume in the noninfarcted myocardium; (3) angiogenesis was activated in the infarcted myocardium, particularly at week 1, and was not altered by treatment with imatinib; and (4) ventricular dysfunction was evident in MI rats at week 4, and mildly improved with imatinib treatment. These observations indicated that PDGF can contribute to the development of cardiac interstitial fibrosis in the noninfarcted myocardium, but does not alter scar formation in the infarcted myocardium. Further, this study suggests the potential therapeutic effects of PDGFR blockade on interstitial fibrosis of the infarcted heart.

CD82 restrains pathological angiogenesis by altering lipid raft clustering and CD44 trafficking in endothelial cells.

BACKGROUND: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. METHODS AND RESULTS: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. CONCLUSIONS: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.

Vascular endothelial growth factor-C: its unrevealed role in fibrogenesis.

Vascular endothelial growth factor (VEGF)-C is a key mediator of lymphangiogenesis. Our recent study shows that VEGF-C/VEGF receptors (VEGFR)-3 are significantly increased in the infarcted rat myocardium, where VEGFR-3 is expressed not only in lymph ducts but also in myofibroblasts, indicating that VEGF-C has an unrevealed role in fibrogenesis during cardiac repair. The current study is to explore the regulation and molecular mechanisms of VEGF-C in fibrogenesis. The potential regulation of VEGF-C on myofibroblast differentiation/growth/migration, collagen degradation/synthesis, and transforming growth factor (TGF)-ß and ERK pathways was detected in cultured cardiac myofibroblasts. Our results showed that VEGF-C significantly increased myofibroblast proliferation, migration, and type I/III collagen production. Matrix metalloproteinase (MMP)-2 and -9 were significantly elevated in the medium of VEGF-C-treated cells, coincident with increased tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Furthermore, VEGF-C activated the TGF-ß1 pathway and ERK phosphorylation, which was significantly suppressed by TGF-ß or ERK blockade. This is the first study indicating that in addition to lymphangiogenesis, VEGF-C is also involved in fibrogenesis through stimulation of myofibroblast proliferation, migration, and collagen synthesis, via activation of the TGF-ß1 and ERK pathways.

Autocrine and paracrine function of Angiotensin 1-7 in tissue repair during hypertension.

BACKGROUND: Angiotensin-converting enzyme 2 (ACE2) cleaves angiotensin (Ang) II to generate Ang1-7, which mediates cellular actions through Mas receptors (MasR). Hypertension is accompanied by high or low circulating AngII levels and cardiac/renal injury. The purpose of this study is to explore (i) whether circulating AngII affects ACE2/MasR expressions in the hypertensive heart and kidney; and (ii) whether Ang1-7 regulates cardiac repair/remodeling responses through MasR during hypertension. METHODS: In the first portion of the study, rats received either an AngII infusion (400ng/kg/min) for 4 weeks, leading to hypertension with high circulating AngII, or an aldosterone (ALDO, 0.75 µg/h) infusion for 4 weeks, leading to hypertension with low/normal circulating AngII. Cardiac and renal ACE2/MasR expressions were examined. We found that cardiac ACE2 was increased and MasR attenuated in both AngII and ALDO groups. However, renal ACE2 and MasR remained unchanged in both AngII- and ALDO-treated animals. RESULTS: In the second portion, rats received AngII infusion with/without MasR antagonist (A779, 1mg/kg/day) for 4 weeks. The roles of MasR blockade in cardiac inflammation, fibrosis, apoptosis, and ventricular function were examined. Chronic AngII infusion caused scattered cardiac injuries, and A779 cotreatment exacerbated cardiac injury, resulting in aggravated inflammatory, fibrogenic, and apoptotic responses compared with the AngII group. Cardiac function, however, was unaltered in the AngII and A779 groups. CONCLUSIONS: ACE2 and MasR expressions in the hypertensive heart and kidney are not regulated by circulating AngII levels. Ang1-7 is involved in multiple repair responses, suggesting that therapeutic strategies aimed at administering Ang1-7 hold potential for the management of cardiac remodeling.

Modification of oxidative stress on gene expression profiling in the rat infarcted heart.

Cardiac oxidative stress is developed following myocardial infarction (MI) particularly in the first week of MI. The influence of reactive oxygen species (ROS) on gene expression profiling and molecular pathways in the infarcted myocardium remains uncertain and is explored in the present study. Rats with MI were treated with or without antioxidants for 1 week. Normal rats served as controls. Cardiac oxidative stress and gene profiling were investigated. Compared to normal hearts, malondialdehyde, a marker of oxidative stress, was significantly increased in the infarcted myocardium, which was significantly suppressed by antioxidants. Microarray assay showed that over a thousand genes were differentially expressed in the infarcted myocardium. Antioxidants significantly altered the expression of 159 genes compared to untreated MI rats. Ingenuity pathway analysis indicated that multiple pathway networks were affected by antioxidants, including those related to cell movement, growth/development, death, and inflammatory/fibrotic responses. IPA further identified that these changes were primarily related to NFκB, p38 MAPK, and ERκ1/2 pathways. Hub genes were identified in the associated gene networks. This study reveals the gene networks associated with cardiac oxidative stress postMI. These observations indicate that ROS regulate various molecular and cellular actions related to cardiac repair/remodeling through multiple gene networks.

Platelet-derived growth factor-D promotes fibrogenesis of cardiac fibroblasts.

Platelet-derived growth factor (PDGF)-D is a newly recognized member of the PDGF family with its role just now being understood. Our previous study shows that PDGF-D and its receptors (PDGFR-ß) are significantly increased in the infarcted heart, where PDGFR-ß is primarily expressed by fibroblasts, indicating the involvement of PDGF-D in the development of cardiac fibrosis. In continuing with these findings, the current study explored the molecular basis of PDGF-D on fibrogenesis. Rat cardiac fibroblasts were isolated and treated with PDGF-D (200 ng/ml medium). The potential regulation of PDGF-D on fibroblast growth, phenotype change, collagen turnover, and the transforming growth factor (TGF)-ß pathway were explored. We found: 1) PDGF-D significantly elevated cardiac fibroblast proliferation, myofibroblast (myoFb) differentiation, and type I collagen secretion; 2) matrix metalloproteinase (MMP)-1, MMP-2, and MMP-9 protein levels were significantly elevated in PDGF-D-treated cells, which were coincident with increased expressions of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2; 3) PDGF-D significantly enhanced TGF-ß1 synthesis, which was eliminated by TGF-ß blockade with small-interfering RNA (siRNA); 4) the stimulatory role of PDGF-D on fibroblast proliferation and collagen synthesis was abolished by TGF-ß blockade; and 5) TGF-ß siRNA treatment significantly suppressed PDGF-D synthesis in fibroblasts. These observations indicate that PDGF-D promotes fibrogenesis through multiple mechanisms. Coelevations of TIMPs and MMPs counterbalance collagen degradation. The profibrogenic role of PDGF-D is mediated through activation of the TGF-ß1 pathway. TGF-ß1 exerts positive feedback on PDGF-D synthesis. These findings suggest the potential therapeutic effect of PDGFR blockade on interstitial fibrosis in the infarcted heart.