Takayuki Shindo

Gene Expression in Fibroblasts and Fibrosis: Involvement in Cardiac Hypertrophy

Abstract— Structural remodeling of the ventricular wall is a key determinant of clinical outcome in heart disease. Such remodeling involves the production and destruction of extracellular matrix proteins, cell proliferation and migration, and apoptotic and necrotic cell death. Cardiac fibroblasts are crucially involved in these processes, producing growth factors and cytokines that act as autocrine and paracrine factors, as well as extracellular matrix proteins and proteinases. Recent studies have shown that the interactions between cardiac fibroblasts and cardiomyocytes are essential for the progression of cardiac remodeling. This review addresses the functional role played by cardiac fibroblasts and the molecular mechanisms that govern their activity during cardiac hypertrophy and remodeling. A particular focus is the recent progress toward our understanding of the transcriptional regulatory mechanisms involved.

Krüppel-like zinc-finger transcription factor KLF5/BTEB2 is a target for angiotensin II signaling and an essential regulator of cardiovascular remodeling.

We recently isolated a Krüppel-like zinc-finger transcription factor 5 (KLF5; also known as BTEB2 and IKLF), which is markedly induced in activated vascular smooth-muscle cells and fibroblasts. Here we describe our analysis of the in vivo function of KLF5 using heterozygous KLF5-knockout mice (Klf5+/−). In response to external stress, Klf5+/− mice showed diminished levels of arterial-wall thickening, angiogenesis, cardiac hypertrophy and interstitial fibrosis. Also, angiotensin II induced expression of KLF5, which in turn activated platelet-derived growth factor-A (PDGF-A) and transforming growth factor-β (TGF-β) expression. In addition, we determined that KLF5 interacted with the retinoic-acid receptor (RAR), that synthetic RAR ligands modulated KLF5 transcriptional activity, and that in vivo administration of RAR ligands affected stress responses in the cardiovascular system in a KLF5-dependent manner. KLF5 thus seems to be a key element linking external stress and cardiovascular remodeling.

ADAMTS-1: a metalloproteinase-disintegrin essential for normal growth, fertility, and organ morphology and function.

A disintegrin and metalloproteinase (ADAM) represents a protein family possessing both metalloproteinase and disintegrin domains. ADAMTS-1, an ADAM family member cloned from cachexigenic colon adenocarcinoma, is unusual in that it contains thrombospondin type I motifs and anchors to the extracellular matrix. To elucidate the biological role of ADAMTS-1, we developed ADAMTS-1-null mice by gene targeting. Targeted disruption of the mouse ADAMTS-1 gene resulted in growth retardation with adipose tissue malformation. Impaired female fertilization accompanied by histological changes in the uterus and ovaries also resulted. Furthermore, ADAMTS-1(-/-) mice demonstrated enlarged renal calices with fibrotic changes from the ureteropelvic junction through the ureter, and abnormal adrenal medullary architecture without capillary formation. ADAMTS-1 thus appears necessary for normal growth, fertility, and organ morphology and function. Moreover, the resemblance of the renal phenotype to human ureteropelvic junction obstruction may provide a clue to the pathogenesis of this common congenital disease.

Cardiac fibroblasts are essential for the adaptive response of the murine heart to pressure overload

Fibroblasts, which are the most numerous cell type in the heart, interact with cardiomyocytes in vitro and affect their function; however, they are considered to play a secondary role in cardiac hypertrophy and failure. Here we have shown that cardiac fibroblasts are essential for the protective and hypertrophic myocardial responses to pressure overload in vivo in mice. Haploinsufficiency of the transcription factor-encoding gene Krüppel-like factor 5 (Klf5) suppressed cardiac fibrosis and hypertrophy elicited by moderate-intensity pressure overload, whereas cardiomyocyte-specific Klf5 deletion did not alter the hypertrophic responses. By contrast, cardiac fibroblast-specific Klf5 deletion ameliorated cardiac hypertrophy and fibrosis, indicating that KLF5 in fibroblasts is important for the response to pressure overload and that cardiac fibroblasts are required for cardiomyocyte hypertrophy. High-intensity pressure overload caused severe heart failure and early death in mice with Klf5-null fibroblasts. KLF5 transactivated Igf1 in cardiac fibroblasts, and IGF-1 subsequently acted in a paracrine fashion to induce hypertrophic responses in cardiomyocytes. Igf1 induction was essential for cardioprotective responses, as administration of a peptide inhibitor of IGF-1 severely exacerbated heart failure induced by high-intensity pressure overload. Thus, cardiac fibroblasts play a pivotal role in the myocardial adaptive response to pressure overload, and this role is partly controlled by KLF5. Modulation of cardiac fibroblast function may provide a novel strategy for treating heart failure, with KLF5 serving as an attractive target.

Evidence for association between paraoxonase gene polymorphisms and atherosclerotic diseases

Paraoxonase 1 (PON1) is proposed to have an anti-atherogenic action. Two polymorphisms at the PON1 (M/L55 and Q/R192) have been shown to be associated with coronary artery disease (CAD). This conclusion is not drawn universally, however, and specific ethnic characteristics may be important determinants in this association. Recently two homologues of PON1 - PON2 and PON3 - were identified and Sanghera et al. demonstrated C/S311 polymorphism at PON2 was associated with the risk of CAD. Within that context, we investigated the association between the aforementioned three polymorphisms and CAD and ischemic stroke in a Japanese population. The study population included 431 control subjects, 210 CAD patients, and 235 ischemic stroke patients. Genotype distributions and allele frequencies of M/L55 and C/S311 were similar among the control and patient groups, whereas the R192 allele frequency was significantly higher (P<0.001) in CAD (75%) and ischemic stroke (76%) patients than in control subjects (65%). When confounding influences of other risk factors were controlled for by multivariate analysis, R192 remained an independent risk determinant (additive model: OR (95% CI), P value CAD: 2.01 (1.45-2.79), 0.0001; ischemic stroke: 1.84 (1.34-2.52), 0.0002 (three genotypes into calculation)). Taken together, our data indicate that the Q/R192 is principally associated with both CAD and ischemic stroke in Japanese.

Hypotension and Resistance to Lipopolysaccharide-Induced Shock in Transgenic Mice Overexpressing Adrenomedullin in Their Vasculature

BACKGROUND Adrenomedullin (AM) is a vasodilating peptide involved in the regulation of circulatory homeostasis and in the pathophysiology of certain cardiovascular diseases. To determine the extent to which chronic AM overproduction affects circulatory physiology under normal and pathological conditions, we used a preproendothelin-1 promoter to establish transgenic mouse lines overexpressing AM in their vasculature. METHODS AND RESULTS Transgenic mice overexpressing AM mainly in vascular endothelial and smooth muscle cells exhibited significantly lower blood pressure (BP) and higher plasma cGMP levels than their wild-type littermates. Blockade of NO synthase with N(G)-monomethyl-L-arginine elevated BP to a greater degree in AM transgenic mice, offsetting the BP difference between the 2 groups. Despite their lower basal BP, administration of bacterial lipopolysaccharide elicited smaller declines in BP and less severe organ damage in AM transgenic mice than in wild-type mice. Furthermore, the 24-hour survival rate after induction of lipopolysaccharide shock was significantly higher in the transgenic mice. CONCLUSIONS A chronic increase in vascular AM production reduces BP at least in part via an NO-dependent pathway. In addition, smaller responses to LPS in transgenic mice suggest that AM is protective against the circulatory collapse, organ damage, and mortality characteristic of endotoxic shock.

The GPCR modulator protein RAMP2 is essential for angiogenesis and vascular integrity

Adrenomedullin (AM) is a peptide involved both in the pathogenesis of cardiovascular diseases and in circulatory homeostasis. The high-affinity AM receptor is composed of receptor activity-modifying protein 2 or 3 (RAMP2 or -3) and the GPCR calcitonin receptor-like receptor. Testing our hypothesis that RAMP2 is a key determinant of the effects of AM on the vasculature, we generated and analyzed mice lacking RAMP2. Similar to AM-/- embryos, RAMP2-/- embryos died in utero at midgestation due to vascular fragility that led to severe edema and hemorrhage. Vascular ECs in RAMP2-/- embryos were severely deformed and detached from the basement membrane. In addition, the abnormally thin arterial walls of these mice had a severe disruption of their typically multilayer structure. Expression of tight junction, adherence junction, and basement membrane molecules by ECs was diminished in RAMP2-/- embryos, leading to paracellular leakage and likely contributing to the severe edema observed. In adult RAMP2+/- mice, reduced RAMP2 expression led to vascular hyperpermeability and impaired neovascularization. Conversely, ECs overexpressing RAMP2 had enhanced capillary formation, firmer tight junctions, and reduced vascular permeability. Our findings in human cells and in mice demonstrate that RAMP2 is a key determinant of the effects of AM on the vasculature and is essential for angiogenesis and vascular integrity.

Methylenetetrahydrofolate Reductase Gene Polymorphism and Ischemic Stroke in Japanese

Hyperhomocyst(e)inemia has been identified as an independent risk factor for atherosclerotic and thromboembolic diseases such as coronary artery disease, cerebral artery disease, and venous thrombosis. Recently, the alanine/valine (A/V) gene polymorphism of 5,10-methylenetetrahydrofolate reductase (MTHFR), one of the key enzymes that catalyzes the remethylation of homocysteine, was reported. The VV genotype is correlated with increased plasma homocyst(e)ine levels as a result of the reduced activity and increased thermolability of this enzyme. In this study, we examined the association between the V allele of the MTHFR gene and ischemic stroke in an elderly Japanese population. The diagnosis of cerebral infarction of all study patients was confirmed by CT of the brain. The MTHFR genotype was analyzed by polymerase chain reaction followed by HinfI digestion. In 256 stroke patients and 325 control subjects, the frequencies of the V allele were 0.45 and 0.32, respectively. The odds ratios and 95% confidence intervals adjusted for the other risk factors were, respectively, 1.51 (1.02 to 2.23) for the AV genotype and 3.35 (1.94 to 5.77) for the VV genotype compared with the AA genotype. Both of these effects were statistically significant (P=0.041 and P<0.001, respectively). In patients with multiple infarcts in particular, the allele frequency of the V mutation was 0.56, and the association between the V allele and stroke was highly significant. Plasma homocyst(e)ine levels were significantly higher in patients with the VV genotype than in patients with the AA or AV genotype, especially those with low plasma folate levels. The V allele of the MTHFR gene was significantly associated with cerebral infarction in an elderly Japanese population in a codominant manner. The VV genotype may contribute to risk for ischemic stroke through a predisposition to increased plasma homocyst(e)ine levels, and dietary folate supplementation may be of benefit, particularly to patients with this genotype.

Angiogenic Effects of Adrenomedullin in Ischemia and Tumor Growth

Adrenomedullin (AM) is a novel vasodilating peptide involved in the regulation of circulatory homeostasis and implicated in the pathophysiology of cardiovascular disease. We tested the hypothesis that AM also possesses angiogenic properties. Using laser Doppler perfusion imaging, we found that AM stimulated recovery of blood flow to the affected limb in the mouse hind-limb ischemia model. AM exerted this effect in part by promoting expression of vascular endothelial growth factor (VEGF) in the ischemic limb, and immunostaining for CD31 showed the enhanced flow to reflect increased collateral capillary density. By enhancing tumor angiogenesis, AM also promoted the growth of subcutaneously transplanted sarcoma 180 tumor cells. However, heterozygotic AM knockout mice (AM+/−) showed significantly less blood flow recovery with less collateral capillary development and VEGF expression than their wild-type littermates. Similarly, mice treated with AM22-52, a competitive inhibitor of AM, showed reduced capillary development, and growth of sarcoma 180 tumors was inhibited in AM+/− and AM22-52–treated mice. Notably, administration of VEGF or AM rescued blood flow recovery and capillary formation in AM+/− and AM22-52–treated mice. In cocultures of endothelial cells and fibroblasts, AM enhanced VEGF-induced capillary formation, whereas in cultures of endothelial cells AM enhanced VEGF-induced Akt activation. These results show that AM possesses novel angiogenic properties mediated by its ability to enhance VEGF expression and Akt activity. This may make AM a useful therapeutic tool for relieving ischemia; conversely, inhibitors of AM could be useful for clinical management of tumor growth.

Protective Effects of Endogenous Adrenomedullin on Cardiac Hypertrophy, Fibrosis, and Renal Damage

BACKGROUND Adrenomedullin (AM) is a novel vasodilating peptide thought to have important effects on cardiovascular function. The aim of this study was to assess the activity of endogenous AM in the cardiovascular system using AM knockout mice. METHODS AND RESULTS Mice heterozygous for an AM-null mutation (AM+/-) and their wild-type littermates were subjected to aortic constriction or angiotensin II (Ang II) infusion. The resultant cardiovascular stress led to increases in heart weight/body weight ratios, left ventricular wall thickness, and perivascular fibrosis, as well as expression of genes encoding angiotensinogen, ACE, transforming growth factor-beta, collagen type I, brain natriuretic peptide, and c-fos. In addition, renal damage characterized by decreased creatinine clearance with glomerular sclerosis was noted. In all cases, the effects were significantly more pronounced in AM+/- mice. Hearts from adult mice subjected to aortic constriction showed enhanced extracellular signal-regulated kinase (ERK) activation, as did cardiac myocytes from neonates treated acutely with Ang II. Again the effect was more pronounced in AM+/- mice, which showed increases in cardiac myocyte size, protein synthesis, and fibroblast proliferation. ERK activation was suppressed by protein kinase C inhibition to a greater degree in AM+/- myocytes. In addition, treatment of cardiac myocytes with recombinant AM suppressed Ang II-induced ERK activation via a protein kinase A-dependent pathway. CONCLUSIONS Endogenous AM exerts a protective effect against stress-induced cardiac hypertrophy via protein kinase C- and protein kinase A-dependent regulation of ERK activation. AM may thus represent a useful new tool for the treatment of cardiovascular disease.