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.

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.

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.

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.

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.

Plant Leaf Functional Adaptions along Urban-Rural Gradients of Jinhua City.

Environmental changes induced by urbanization may significantly alter plant survival strategies, thereby introducing uncertainties in their ability to withstand extreme heat. This study, centered on Jinhua City, distinguished urban, suburban, and rural areas to represent the various intensities of urbanization. It examined the leaf function properties of evergreen and deciduous trees common in these regions, focusing on leaf and branch characteristics. Employing an analysis of variance (ANOVA), principal component analysis (PCA), and path analysis (PA) of the plant functional traits and the climatic factors of each region, this study assessed the impact of urbanization on plant survival strategies. By tracking changes in plant functional traits from June to August, it explored the capacity of plants to acclimate to urban-warming-related heat stress across different urbanization gradients. The findings revealed that leaf thickness (LT) and stomatal size (SS) initially decreased and then increased, whereas specific leaf area (SLA) and leaf tissue density (LTD) first rose and then declined, from rural to urban regions. From June to August, branch wood density (WD), chlorophyll (Chl) content, LTD, and leaf dry matter content (LDMC) increased, whereas SLA and leaf water content (LWC) diminished, in all regions. PCA suggested that there was no significant change in the resource allocation strategy of plants (p > 0.05), with drought tolerance significantly reduced in the suburbs on the gradient of urbanization (p < 0.05). During the summer, with high temperature, plants were predominantly biased towards slow-return, conservative strategies, particularly among evergreen species. Compared to precipitation, PA revealed a significant urban warming effect. During summer, temperature was the main factor influencing resource investment strategy and drought resistance, with a notably stronger impact on the former. The high temperature in summer promoted a conservative survival strategy in plants, and the urbanization effect increased their tolerance to high temperatures.

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.

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.

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.

Platelet-derived growth factor involvement in myocardial remodeling following infarction.

Cardiac remodeling occurs in the infarcted heart (MI). The underlying regulatory mechanisms are under investigation. Platelet-derived growth factor (PDGF) is a family of growth factors that stimulates cell growth, differentiation and migration. Herein, we sought to determine whether PDGF is involved in cardiac repair/remodeling following MI. The temporal and spatial expressions of PDGF isoforms (A, B, C and D) and PDGF receptor (PDGFR)-α and ß as well as cell types expressing PDGF were examined in the infarcted rat heart. Sham-operated rats served as controls. We found that the normal myocardium expressed all PDGF isoforms, and cell types expressing PDGF were primarily interstitial cells. Following MI, PDGF-A and D were significantly increased in the infarcted myocardium during 6 weeks of the observation period and cells expressing PDGF-A and D were primarily endothelial cells, macrophages and myofibroblasts (myoFb). PDGF-B and C expressions were, however, reduced in the infarcted heart. In the noninfarcted myocardium, PDGF-D expression was increased in the late stage of MI and cells expressing PDGF-D were predominantly fibroblasts. Both PDGFR-α and ß were significantly increased in the infarcted myocardium in the early and late stages of MI and in the noninfarcted myocardium in the late stage of MI. Enhanced PDGF-A, PDGF-D and PDGFR are coincident with angiogenesis, and inflammatory and fibrogenic responses in the infarcted myocardium, suggesting their regulation on cardiac repair. Elevated PDGF-D in the noninfarcted myocardium suggests its involvement in the development of interstitial fibrosis that appears in the late stage of MI.

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.

Vascular endothelial growth factor (VEGF)-A: role on cardiac angiogenesis following myocardial infarction.

The current study is to determine the regulatory role of VEGF-A in cardiac angiogenesis following myocardial infarction (MI). Cardiac angiogenic response and temporal/spatial expression of VEGF-A/VEGF receptors (VEGFR) were examined at 1, 2, 6, 12 h and 1, 2, 3, 4, 7, 14, and 28 days postMI. We found that following MI, newly formed vessels first appeared at the border zone between noninfarcted and infarcted myocardium as early as day 3 and subsequently in the infarcted myocardium. Vascular density in the infarcted myocardium peaked at day 7 and then gradually declined. VEGF-A mRNA started to increase at the border zone at 2 h postMI, reached peak at 12 h, declined at day 1, and returned to normal levels at day 2 and thereafter. VEGF-A protein levels at the border zone were only increased during day 1 postMI. VEGF-A within the infarcted myocardium levels, however, was persistently suppressed postMI. VEGFR expression was significantly increased only at the border zone at day 1, but not in the later stages. The expression of VEGF-A/VEGFR remained unchanged in the noninfarcted myocardium. Thus, the early rise of VEGF-A/VEGFR at the border zone suggests that VEGF-A initiates the cardiac angiogenic response postMI, but short-lived VEGF-A/VEGFR activation at the border zone and consistently suppressed VEGF-A within the infarcted myocardium suggests that VEGF-A may not be crucial to the later stages of angiogenesis.

Reactive oxygen species promote angiogenesis in the infarcted rat heart.

The purpose of this study was to determine whether reactive oxygen species (ROS) promote cardiac angiogenesis following myocardial infarction (MI) and contribute to cardiac repair. Rats with MI were treated with or without antioxidants, tempol and apocynin. Hearts of these rats were collected at days 2, 4, 7 and 14 post-MI. We examined the spatial and temporal relationship between oxidative stress and angiogenesis as well as the potential regulation of ROS in cardiac angiogenesis. We found: (i) following MI, gp91(phox), a subunit of NADPH oxidase, a key enzyme for ROS production, was significantly increased in the border zone at day 2, followed by the infarcted myocardium at day 4, peaked at day 7 and declined at day 14, while superoxide dismutase was significantly reduced; (ii) malondialdehyde, a marker of oxidative stress, was significantly increased in the infarcted myocardium at day 7; (iii) pre-existing blood vessels in the infarcted myocardium underwent necrosis post-MI, whereas newly formed vessels appeared at the border zone at day 4, and then extended into the infarcted myocardium, where microvascular density peaked at day 7 and (iv) antioxidant treatment significantly reduced microvascular density in the infarcted myocardium at day 7. These observations suggest that following MI, angiogenesis is mostly active in the infarcted myocardium in the first week, which is temporally and spatially coincident with enhanced ROS. Suppression of angiogenesis by antioxidants indicates that ROS promote angiogenesis in the infarcted myocardium and contribute to cardiac repair. Further studies are required to determine the mechanisms responsible for ROS-mediated cardiac angiogenesis.

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.

The microenvironmental determinants for kidney epithelial cyst morphogenesis.

Although epithelial morphogenesis is tightly controlled by intrinsic genetic programs, the microenvironment in which epithelial cells proliferate and differentiate also contributes to the morphogenetic process. The roles of the physical microenvironment in epithelial morphogenesis, however, have not been well dissected. In this study, we assessed the impact of the microenvironment on epithelial cyst formation, which often marks the beginning or end step of morphogenesis of epithelial tissues and the pathological characteristic of some diseases. Previous studies have demonstrated that Madin-Darby canine kidney (MDCK) epithelial cells form cysts when grown in a three-dimensional (3D) extracellullar matrix (ECM) environment. We have now further demonstrated that the presence of ECM in the 3D scaffold is required for the formation of properly polarized cysts. Also, we have found that the full interface of epithelial cells with the ECM environment (in-3D) is not essential for cyst formation, since partial contact (on-3D) is sufficient to induce cystogenesis. In addition, we have defined the minimal ECM environment or the physical threshold for cystogenesis under the on-3D condition. Only above the threshold can the morphological cues from the ECM environment induce cyst formation. Moreover, cyst formation under the on-3D condition described in this study defines a novel and more feasible model to analyze in vitro morphogenesis. Finally, we have found that, during cystogenesis, MDCK cells generate basal microprotrusions and produce vesicle-like structures to the basal extracellular space, which are specific to and correlated with cyst formation. For the first time, we have systematically and quantitatively elucidated the microenvironmental determinants for epithelial cystogenesis.

Kidney fibrosis in hypertensive rats: role of oxidative stress.

Fibrosis of the glomerulus and the tubulointerstitium occurs in patients with hypertension. Studies have shown that renal oxidative stress appears in hypertensive kidney disease. The potential role of oxidative stress in renal fibrogenesis remains to be elucidated. Herein, we tested the hypothesis that oxidative stress contributes to the development of renal fibrosis during hypertension.Sprague-Dawley rats received angiotensin II (AngII; 9 microg/h s.c.) for 4 weeks with/without co-treatment of antioxidants, apocynin and tempol (120 mg/kg/day each, p.o.). Untreated rats served as controls. Appearance of renal oxidative stress and its effect on the expression of transforming growth factor (TGF)-beta(1), population of myofibroblasts, collagen synthesis/degradation and fibrosis in kidneys were examined. Chronic AngII infusion elevated systemic blood pressure (228 +/- 6 mm Hg), which was accompanied with extensive renal fibrosis and oxidative stress represented as upregulated NADPH oxidase and suppressed superoxide dismutase (SOD). Co-treatment with antioxidants led to: (1) markedly decreased renal NADPH oxidase; (2) significantly attenuated gene expression of TGF-beta(1), type I collagen, and tissue inhibitors of matrix metalloproteinase (TIMP)-I/-II in the kidney; (3) largely reduced population of myofibroblasts in both the cortex and medulla; (4) significantly reduced renal collagen volume, and (5) partially suppressed blood pressure (190 +/- 8 mm Hg). Thus, prolonged AngII administration promotes renal oxidative stress, which is associated with hypertensive renal disease. AngII induces renal oxidative stress by increasing NADPH oxidase and reducing SOD in the kidney, which, in turn, upregulates collagen synthesis, while suppressing collagen degradation, thereby promoting the development of fibrosis in kidneys of hypertensive rats.

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.