Loss of MURC/Cavin-4 induces JNK and MMP-9 activity enhancement in vascular smooth muscle cells and exacerbates abdominal aortic aneurysm.

Abdominal aortic aneurysm (AAA) is relatively common in elderly patients with atherosclerosis. MURC (muscle-restricted coiled-coil protein)/Cavin-4 modulating the caveolae function of muscle cells is expressed in cardiomyocytes, skeletal muscle cells and smooth muscle cells. Here, we show a novel functional role of MURC/Cavin-4 in vascular smooth muscle cells (VSMCs) and AAA development. Both wild-type (WT) and MURC/Cavin-4 knockout (MURC-/-) mice subjected to periaortic application of CaCl2 developed AAAs. Six weeks after CaCl2 treatment, internal and external aortic diameters were significantly increased in MURC-/- AAAs compared with WT AAAs, which were accompanied by advanced fibrosis in the tunica media of MURC-/- AAAs. The activity of JNK and matrix metalloproteinase (MMP) -2 and -9 were increased in MURC-/- AAAs compared with WT AAAs at 5 days after CaCl2 treatment. At 6 weeks after CaCl2 treatment, MURC-/- AAAs exhibited attenuated JNK activity compared with WT AAAs. There was no difference in the activity of MMP-2 or -9 between saline and CaCl2 treatments. In MURC/Cavin-4-knockdown VSMCs, TNFα-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Furthermore, WT, MURC-/-, apolipoprotein E-/- (ApoE-/-), and MURC/Cavin-4 and ApoE double-knockout (MURC-/-ApoE-/-) mice were subjected to angiotensin II (Ang II) infusion. In both ApoE-/- and MURC-/-ApoE-/- mice infused for 4 weeks with Ang II, AAAs were promoted. The internal aortic diameter was significantly increased in Ang II-infused MURC-/-ApoE-/- mice compared with Ang II-infused ApoE-/- mice. In MURC/Cavin-4-knockdown VSMCs, Ang II-induced activity of JNK and MMP-9 was enhanced compared with control VSMCs. Our results suggest that MURC/Cavin-4 in VSMCs modulates AAA progression at the early stage via the activation of JNK and MMP-9. MURC/Cavin-4 is a potential therapeutic target against AAA progression.

MURC/Cavin-4 facilitates recruitment of ERK to caveolae and concentric cardiac hypertrophy induced by α1-adrenergic receptors.

The actions of catecholamines on adrenergic receptors (ARs) induce sympathetic responses, and sustained activation of the sympathetic nervous system results in disrupted circulatory homeostasis. In cardiomyocytes, α1-ARs localize to flask-shaped membrane microdomains known as "caveolae." Caveolae require both caveolin and cavin proteins for their biogenesis and function. However, the functional roles and molecular interactions of caveolar components in cardiomyocytes are poorly understood. Here, we showed that muscle-restricted coiled-coil protein (MURC)/Cavin-4 regulated α1-AR-induced cardiomyocyte hypertrophy through enhancement of ERK1/2 activation in caveolae. MURC/Cavin-4 was expressed in the caveolae and T tubules of cardiomyocytes. MURC/Cavin-4 overexpression distended the caveolae, whereas MURC/Cavin-4 was not essential for their formation. MURC/Cavin-4 deficiency attenuated cardiac hypertrophy induced by α1-AR stimulation in the presence of caveolae. Interestingly, MURC/Cavin-4 bound to α1A- and α1B-ARs as well as ERK1/2 in caveolae, and spatiotemporally modulated MEK/ERK signaling in response to α1-AR stimulation. Thus, MURC/Cavin-4 facilitates ERK1/2 recruitment to caveolae and efficient α1-AR signaling mediated by caveolae in cardiomyocytes, which provides a unique insight into the molecular mechanisms underlying caveola-mediated signaling in cardiac hypertrophy.

Inhibition of p53 preserves Parkin-mediated mitophagy and pancreatic ß-cell function in diabetes.

Mitochondrial compromise is a fundamental contributor to pancreatic ß-cell failure in diabetes. Previous studies have demonstrated a broader role for tumor suppressor p53 that extends to the modulation of mitochondrial homeostasis. However, the role of islet p53 in glucose homeostasis has not yet been evaluated. Here we show that p53 deficiency protects against the development of diabetes in streptozotocin (STZ)-induced type 1 and db/db mouse models of type 2 diabetes. Glucolipotoxicity stimulates NADPH oxidase via receptor for advanced-glycation end products and Toll-like receptor 4. This oxidative stress induces the accumulation of p53 in the cytosolic compartment of pancreatic ß-cells in concert with endoplasmic reticulum stress. Cytosolic p53 disturbs the process of mitophagy through an inhibitory interaction with Parkin and induces mitochondrial dysfunction. The occurrence of mitophagy is maintained in STZ-treated p53(-/-) mice that exhibit preserved glucose oxidation capacity and subsequent insulin secretion signaling, leading to better glucose tolerance. These protective effects are not observed when Parkin is deleted. Furthermore, pifithrin-α, a specific inhibitor of p53, ameliorates mitochondrial dysfunction and glucose intolerance in both STZ-treated and db/db mice. Thus, an intervention with cytosolic p53 for a mitophagy deficiency may be a therapeutic strategy for the prevention and treatment of diabetes.

Loss of apoptosis regulator through modulating IAP expression (ARIA) protects blood vessels from atherosclerosis.

Atherosclerosis is the primary cause for cardiovascular disease. Here we identified a novel mechanism underlying atherosclerosis, which is provided by ARIA (apoptosis regulator through modulating IAP expression), the transmembrane protein that we recently identified. ARIA is expressed in macrophages present in human atherosclerotic plaque as well as in mouse peritoneal macrophages. When challenged with acetylated LDL, peritoneal macrophages isolated from ARIA-deficient mice showed substantially reduced foam cell formation, whereas the uptake did not differ from that in wild-type macrophages. Mechanistically, loss of ARIA enhanced PI3K/Akt signaling and consequently reduced the expression of acyl coenzyme A:cholesterol acyltransferase-1 (ACAT-1), an enzyme that esterifies cholesterol and promotes its storage, in macrophages. Inhibition of PI3K abolished the reduction in ACAT-1 expression and foam cell formation in ARIA-deficient macrophages. In contrast, overexpression of ARIA reduced Akt activity and enhanced foam cell formation in RAW264.7 macrophages, which was abrogated by treatment with ACAT inhibitor. Of note, genetic deletion of ARIA significantly reduced the atherosclerosis in ApoE-deficient mice. Oil red-O-positive lipid-rich lesion was reduced, which was accompanied by an increase of collagen fiber and decrease of necrotic core lesion in atherosclerotic plaque in ARIA/ApoE double-deficient mice. Analysis of bone marrow chimeric mice revealed that loss of ARIA in bone marrow cells was sufficient to reduce the atherosclerogenesis in ApoE-deficient mice. Together, we identified a unique role of ARIA in the pathogenesis of atherosclerosis at least partly by modulating macrophage foam cell formation. Our results indicate that ARIA could serve as a novel pharmacotherapeutic target for the treatment of atherosclerotic diseases.

Maternal high-fat diet exaggerates atherosclerosis in adult offspring by augmenting periaortic adipose tissue-specific proinflammatory response.

OBJECTIVE: Maternal obesity elicits offspring's metabolic disorders via developmental modifications of visceral adipose tissue; however, its effect on atherogenesis remains undefined. Perivascular adipose tissue has recently been implicated in vascular remodeling and vasoreactivity. We hypothesize that developmental modifications of perivascular adipose tissue by maternal high-fat diet (HFD) exposure promotes atherosclerosis in adult offspring. APPROACH AND RESULTS: Eight-week-old female apolipoprotein E-deficient mice were fed an HFD or normal diet (ND) during gestation and lactation. Offspring were fed a high-cholesterol diet from 8 weeks of age. Twenty-week-old male offspring of HFD-fed dams (O-HFD) showed a 2.1-fold increase in atherosclerotic lesion of the entire aorta compared with those of ND-fed dams (O-ND). Although mRNA expressions of interleukin-6, tumor necrosis factor, and monocyte chemotactic protein-1 and accumulation of macrophages in epididymal white adipose tissue were less in O-HFD than in O-ND, thoracic periaortic adipose tissue (tPAT) showed an exaggerated inflammatory response in O-HFD. Intra-abdominal transplantation of tPAT from 8-week-old O-HFD alongside the distal abdominal aorta exaggerated atherosclerosis development of the infrarenal aorta in recipient apolipoprotein E-deficient mice compared with tPAT from O-ND (210%, P<0.01). Although macrophage accumulation was rarely detected in tPAT of 8-week-old offspring, mRNA expression and protein levels of macrophage colony-stimulating factor were markedly elevated in O-HFD (2.3-fold, 3.3-fold, respectively, P<0.05), suggesting that increased macrophage colony-stimulating factor expression contributes to the augmented accumulation of macrophages, followed by the enhanced proinflammatory response. CONCLUSIONS: Our findings demonstrate that maternal HFD exaggerates atherosclerosis development in offspring by augmenting tPAT-specific inflammatory response proceeded by an increased expression of macrophage colony-stimulating factor.

Manipulation of cardiac phosphatidylinositol 3-kinase (PI3K)/Akt signaling by apoptosis regulator through modulating IAP expression (ARIA) regulates cardiomyocyte death during doxorubicin-induced cardiomyopathy.

PI3K/Akt signaling plays an important role in the regulation of cardiomyocyte death machinery, which can cause stress-induced cardiac dysfunction. Here, we report that apoptosis regulator through modulating IAP expression (ARIA), a recently identified transmembrane protein, regulates the cardiac PI3K/Akt signaling and thus modifies the progression of doxorubicin (DOX)-induced cardiomyopathy. ARIA is highly expressed in the mouse heart relative to other tissues, and it is also expressed in isolated rat cardiomyocytes. The stable expression of ARIA in H9c2 cardiac muscle cells increased the levels of membrane-associated PTEN and subsequently reduced the PI3K/Akt signaling and the downstream phosphorylation of Bad, a proapoptotic BH3-only protein. When challenged with DOX, ARIA-expressing H9c2 cells exhibited enhanced apoptosis, which was reversed by the siRNA-mediated silencing of Bad. ARIA-deficient mice exhibited normal heart morphology and function. However, DOX-induced cardiac dysfunction was significantly ameliorated in conjunction with reduced cardiomyocyte death and cardiac fibrosis in ARIA-deficient mice. Phosphorylation of Akt and Bad was substantially enhanced in the heart of ARIA-deficient mice even after treatment with DOX. Moreover, repressing the PI3K by cardiomyocyte-specific expression of dominant-negative PI3K (p110α) abolished the cardioprotective effects of ARIA deletion. Notably, targeted activation of ARIA in cardiomyocytes but not in endothelial cells reduced the cardiac PI3K/Akt signaling and exacerbated the DOX-induced cardiac dysfunction. These studies, therefore, revealed a previously undescribed mode of manipulating cardiac PI3K/Akt signaling by ARIA, thus identifying ARIA as an attractive new target for the prevention of stress-induced myocardial dysfunction.

The coiled-coil domain of MURC/cavin-4 is involved in membrane trafficking of caveolin-3 in cardiomyocytes.

Muscle-restricted coiled-coil protein (MURC), also referred to as cavin-4, is a member of the cavin family that works cooperatively with caveolins in caveola formation and function. Cavins are cytoplasmic proteins with coiled-coil domains and form heteromeric complexes, which are recruited to caveolae in cells expressing caveolins. Among caveolins, caveolin-3 (Cav3) is exclusively expressed in muscle cells, similar to MURC/cavin-4. In the heart, Cav3 overexpression contributes to cardiac protection, and its deficiency leads to progressive cardiomyopathy. Mutations in the MURC/cavin-4 gene have been identified in patients with dilated cardiomyopathy. In the present study, we show the role of MURC/cavin-4 as a caveolar component in the heart. In H9c2 cells, MURC/cavin-4 was localized at the plasma membrane, whereas a MURC/cavin-4 mutant lacking the coiled-coil domain (ΔCC) was primarily localized to the cytoplasm. ΔCC bound to Cav3 and impaired membrane localization of Cav3 in cardiomyocytes. Additionally, although ΔCC did not alter Cav3 mRNA expression, ΔCC decreased the Cav3 protein level. MURC/cavin-4 and ΔCC similarly induced cardiomyocyte hypertrophy; however, ΔCC showed higher hypertrophy-related fetal gene expression than MURC/cavin-4. ΔCC induced ERK activation in cardiomyocytes. Transgenic mice expressing ΔCC in the heart (ΔCC-Tg mice) showed impaired cardiac function accompanied by cardiomyocyte hypertrophy and marked interstitial fibrosis. Hearts from ΔCC-Tg mice showed a reduction of the Cav3 protein level and activation of ERK. These results suggest that MURC/cavin-4 requires its coiled-coil domain to target the plasma membrane and to stabilize Cav3 at the plasma membrane of cardiomyocytes and that MURC/cavin-4 functions as a crucial caveolar component to regulate cardiac function.

Pyk2 aggravates hypoxia-induced pulmonary hypertension by activating HIF-1α.

Pulmonary arterial hypertension (PAH) is a refractory disease characterized by uncontrolled vascular remodeling and elevated pulmonary arterial pressure. Although synthetic inhibitors of some tyrosine kinases have been used to treat PAH, their therapeutic efficacies and safeties remain controversial. Thus, the establishment of novel therapeutic targets based on the molecular pathogenesis underlying PAH is a clinically urgent issue. In the present study, we demonstrated that proline-rich tyrosine kinase 2 (Pyk2), a nonreceptor type protein tyrosine kinase, plays a crucial role in the pathogenesis of pulmonary hypertension (PH) using an animal model of hypoxia-induced PH. Resistance to hypoxia-induced PH was markedly higher in Pyk2-deficient mice than in wild-type mice. Pathological investigations revealed that medial thickening of the pulmonary arterioles, which is a characteristic of hypoxia-induced PH, was absent in Pyk2-deficient mice, suggesting that Pyk2 is involved in the hypoxia-induced aberrant proliferation of vascular smooth muscle cells in hypoxia-induced PH. In vitro experiments using human pulmonary smooth muscle cells showed that hypoxic stress increased the proliferation and migration of cells in a Pyk2-dependent manner. We also demonstrated that Pyk2 plays a crucial role in ROS generation during hypoxic stress and that this Pyk2-dependent generation of ROS is necessary for the activation of hypoxia-inducible factor-1α, a key molecule in the pathogenesis of hypoxia-induced PH. In summary, the results of the present study reveal that Pyk2 plays an important role in the pathogenesis of hypoxia-induced PH. Therefore, Pyk2 may represent a promising therapeutic target for PAH in a clinical setting.

Aging differentially alters the expression of angiogenic genes in a tissue-dependent manner.

Organ functions are altered and impaired during aging, thereby resulting in increased morbidity of age-related diseases such as Alzheimer's disease, diabetes, and heart failure in the elderly. Angiogenesis plays a crucial role in the maintenance of tissue homeostasis, and aging is known to reduce the angiogenic capacity in many tissues. Here, we report the differential effects of aging on the expression of angiogenic factors in different tissues, representing a potentially causes for age-related metabolic disorders. PCR-array analysis revealed that many of angiogenic genes were down-regulated in the white adipose tissue (WAT) of aged mice, whereas they were largely up-regulated in the skeletal muscle (SM) of aged mice compared to that in young mice. Consistently, blood vessel density was substantially reduced and hypoxia was exacerbated in WAT of aged mice compared to that in young mice. In contrast, blood vessel density in SM of aged mice was well preserved and was not different from that in young mice. Moreover, we identified that endoplasmic reticulum (ER) stress was strongly induced in both WAT and SM during aging in vivo. We also found that ER stress significantly reduced the expression of angiogenic genes in 3T3-L1 adipocytes, whereas it increased their expression in C2C12 myotubes in vitro. These results collectively indicate that aging differentially affects the expression of angiogenic genes in different tissues, and that aging-associated down-regulation of angiogenic genes in WAT, at least in part through ER stress, is potentially involved in the age-related adipose tissue dysfunction.

Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells.

Endothelial and endothelial progenitor cells (ECs and EPCs) play a fundamental role in angiogenesis that is essential for numerous physiological and pathological processes. The phosphatase and tensin homolog (PTEN)/ phosphoinositide 3-kinase (PI3K) pathway has been implicated in angiogenesis, but the mechanism in the regulation of this pathway in ECs and EPCs is poorly understood. Here we show that ARIA (apoptosis regulator through modulating IAP expression), a transmembrane protein that we recently identified, regulates the PTEN/PI3K pathway in ECs and EPCs and controls developmental and postnatal angiogenesis in vivo. We found that ARIA is abundantly expressed in EPCs and regulates their angiogenic functions by modulating PI3K/Akt/endothelial nitric oxide synthase (eNOS) signaling. Genetic deletion of ARIA caused nonfatal bleeding during embryogenesis, in association with increased small vessel density and altered expression of various vascular growth factors including angiopoietins and VEGF receptors. Postnatal neovascularization induced by critical limb ischemia was substantially enhanced in ARIA-null mice, in conjunction with more bone marrow (BM)-derived ECs detected in ischemic muscles. Administration of PI3K or NO synthase inhibitor completely abolished the enhanced neovascularization in ARIA(-/-) mice. Mechanistically, we identified that ARIA interacts with PTEN at the intracellular domain independently of the PTEN phosphorylation in its C-terminal tail. Overexpressed ARIA increased PTEN in the membrane fraction, whereas ARIA-silencing reduced the membrane-associated PTEN, resulting in modified PI3K/Akt signaling. Taken together, our findings establish a previously undescribed mode of regulation of the PTEN/PI3K/Akt pathway by ARIA, and reveal a unique mechanism in the control of angiogenesis. These functions of ARIA might offer a unique therapeutic potential.

Periaortic adipose tissue-specific activation of the renin-angiotensin system contributes to atherosclerosis development in uninephrectomized apoE-/- mice.

Chronic kidney disease (CKD) is an independent risk factor for the development of cardiovascular disease. The perivascular adipose tissue is closely implicated in the development of atherosclerosis; however, the contribution to CKD-associated atherogenesis remains undefined. Eight-week-old apoE-deficient mice were uninephrectomized and fed a high-cholesterol diet starting at 12 wk of age. The atherosclerotic lesion area in the thoracic aorta was comparable in 16-wk-old uninephrectomized (UNX) mice and sham control mice; however, the lesion area was markedly exaggerated in 20-wk-old UNX mice compared with the control (54%, P < 0.05). While the accumulation of monocytes/macrophages and the mRNA expression levels of inflammatory cytokines/chemokines in the thoracic periaortic adipose tissue (PAT) did not differ between the two groups, angiotensinogen (AGT) mRNA expression and the angiotensin II (ANG II) concentration in the PAT were significantly higher in 16-wk-old UNX mice than in the control (1.9- and 1.5-fold increases vs. control, respectively; P < 0.05). ANG II concentrations in both the plasma and epididymal white adipose tissue (WAT) were comparable between the two groups, suggesting that PAT-specific activation of the renin-angiotensin system (RAS) is primarily involved in CKD-associated atherogenesis. The homeostasis model assessment-insulin resistance (HOMA-IR) index and plasma insulin level after glucose loading were significantly elevated in 16-wk-old UNX mice. In vitro stimulation of preadipocytes with insulin exaggerated the AGT mRNA expression along with increased mRNA expression of PPARγ. These findings suggest that PAT-specific RAS activation probably primarily contributes in accelerating atherosclerotic development in UNX mice and could thus represent a therapeutic target for preventing CKD-associated atherogenesis.

Serglycin is a novel adipocytokine highly expressed in epicardial adipose tissue.

Much recent work has highlighted the key role of adipose tissue as an endocrine organ that secretes a number of adipocytokines, linking adiposity, especially intra-abdominal visceral fat, and the pathogenesis of cardiovascular and metabolic diseases. However, the role of epicardial adipose tissue (EAT), another important visceral fat depot situated in close proximity to epicardial coronary arteries and myocardium, has been less well studied. In this study, we sought to characterize EAT by comparing gene expression profiles of EAT, omental adipose tissue (OAT), and subcutaneous adipose tissue (SCAT) in patients who underwent elective coronary artery bypass graft surgery for critical coronary artery disease (CAD) and identify molecules involved in inflammation. A total of 15,304 probes were detected in all depots, and 231 probes were differentially expressed. Significantly higher expression of pro-inflammatory genes such as interleukin-1ß, -6, and -8, and chemokine receptor 2 was observed in EAT, even when compared with OAT. Among them, serglycin was one of the most abundantly expressed genes in EAT. Serglycin expression was induced during adipocytic differentiation of 3T3L1 cells. Serglycin was secreted from adipocytes, and tumor necrosis factor-α stimulated its expression and secretion in adipocytes. Serglycin was also present in human serum samples. These results suggest that human EAT has strong inflammatory properties in patients with CAD and provide novel evidence that serglycin is an adipocytokine highly expressed in EAT.

p53-TIGAR axis attenuates mitophagy to exacerbate cardiac damage after ischemia.

Inhibition of tumor suppressor p53 is cardioprotective against ischemic injury and provides resistance to subsequent cardiac remodeling. We investigated p53-mediated expansion of ischemic damage with a focus on mitochondrial integrity in association with autophagy and apoptosis. p53(-/-) heart showed that autophagic flux was promoted under ischemia without a change in cardiac tissue ATP content. Electron micrographs revealed that ischemic border zone in p53(-/-) mice had 5-fold greater numbers of autophagic vacuoles containing mitochondria, indicating the occurrence of mitophagy, with an apparent reduction of abnormal mitochondria compared with those in WT mice. Analysis of autophagic mediators acting downstream of p53 revealed that TIGAR (TP53-induced glycolysis and apoptosis regulator) was exclusively up-regulated in ischemic myocardium. TIGAR(-/-) mice exhibited the promotion of mitophagy followed by decrease of abnormal mitochondria and resistance to ischemic injury, consistent with the phenotype of p53(-/-) mice. In p53(-/-) and TIGAR(-/-) ischemic myocardium, ROS production was elevated and followed by Bnip3 activation which is an initiator of mitophagy. Furthermore, the activation of Bnip3 and mitophagy due to p53/TIGAR inhibition were reversed with antioxidant N-acetyl-cysteine, indicating that this adaptive response requires ROS signal. Inhibition of mitophagy using chloroquine in p53(-/-) or TIGAR(-/-) mice exacerbated accumulation of damaged mitochondria to the level of wild-type mice and attenuated cardioprotective action. These findings indicate that p53/TIGAR-mediated inhibition of myocyte mitophagy is responsible for impairment of mitochondrial integrity and subsequent apoptosis, the process of which is closely involved in p53-mediated ventricular remodeling after myocardial infarction.

NFAT5 regulates the canonical Wnt pathway and is required for cardiomyogenic differentiation.

While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expression was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2'Z, 3'E]-6-bromoindirubin-3'-oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.

PARM-1 promotes cardiomyogenic differentiation through regulating the BMP/Smad signaling pathway.

PARM-1, prostatic androgen repressed message-1, is an endoplasmic reticulum (ER) molecule that is involved in ER stress-induced apoptosis in cardiomyocytes. In this study, we assessed whether PARM-1 plays a role in the differentiation of stem cells into cardiomyocytes. While PARM-1 was not expressed in undifferentiated P19CL6 embryonic carcinoma cells, PARM-1 expression was induced during cardiomyogenic differentiation. This expression followed expression of mesodermal markers, and preceded expression of cardiac transcription factors. PARM-1 overexpression did not alter the expression of undifferentiated markers and the proliferative property in undifferentiated P19CL6 cells. Expression of cardiac transcription factors during cardiomyogenesis was markedly enhanced by overexpression of PARM-1, while expression of mesodermal markers was not altered, suggesting that PARM-1 is involved in the differentiation from the mesodermal lineage to cardiomyocytes. Furthermore, overexpression of PARM-1 induced BMP2 mRNA expression in undifferentiated P19CL6 cells and enhanced both BMP2 and BMP4 mRNA expression in the early phase of cardiomyogenesis. PARM-1 overexpression also enhanced phosphorylation of Smads1/5/8. Thus, PARM-1 plays an important role in the cardiomyogenic differentiation of P19CL6 cells through regulating BMP/Smad signaling pathways, demonstrating a novel role of PARM-1 in the cardiomyogenic differentiation of stem cells.

Macrophages play a unique role in the plaque calcification by enhancing the osteogenic signals exerted by vascular smooth muscle cells.

Vascular calcification is a major risk factor for the cardiovascular disease, yet its underlying molecular mechanisms remain to be elucidated. Recently, we identified that osteogenic signals via bone morphogenetic protein (BMP)-2 exerted by vascular smooth muscle cells (VSMCs) play a crucial role in the formation of atherosclerotic plaque calcification. Here we report a synergistic interaction between macrophages and VSMCs with respect to plaque calcification. Treatment with conditioned medium (CM) of macrophages dramatically enhanced BMP-2 expression in VSMCs, while it substantially reduced the expression of matrix Gla-protein (MGP) that inhibits the BMP-2 osteogenic signaling. As a result, macrophages significantly accelerated the osteoblastic differentiation of C2C12 cells induced by VSMC-CM. In contrast, macrophage-CM did not enhance the osteoblastic gene expressions in VSMCs, indicating that macrophages unlikely induced the osteoblastic trans-differentiation of VSMCs. We then examined the effect of recombinant TNF-α and IL-1ß on the VSMC-derived osteogenic signals. Similar to the macrophage-CM, both cytokines enhanced BMP-2 expression and reduced MGP expression in VSMCs. Nevertheless, only the neutralization of TNF-α but not IL-1ß attenuated the effect of macrophage-CM on the expression of these genes in VSMCs, due to the very low concentration of IL-1ß in the macrophage-CM. On the other hand, VSMCs significantly enhanced IL-1ß expression in macrophages, which might in turn accelerate the VSMC-mediated osteogenic signals. Together, we identified a unique role of macrophages in the formation of plaque calcification in coordination with VSMCs. This interaction between macrophages and VSMCs is a potential therapeutic target to treat and prevent the atherosclerotic plaque calcification.

Early inflammatory reactions in atherosclerosis are induced by proline-rich tyrosine kinase/reactive oxygen species-mediated release of tumor necrosis factor-alpha and subsequent activation of the p21Cip1/Ets-1/p300 system.

OBJECTIVE: Reactive oxygen species (ROS) are involved in the initial process of atherosclerosis, whereas it remains to be determined how atherogenic stimulus causes ROS-mediated proinflammatory reactions. Here, we focused on proline-rich tyrosine kinase (PYK2)-mediated ROS generation and examined how atherogenic stimulus causes early proinflammatory reactions. METHODS AND RESULTS: PYK2-deficient (knockout [KO]) (PYK2-KO) mice were crossbred with apolipoprotein E (ApoE)-deficient (PYK2-KO/ApoE-KO) mice. PYK2-KO/ApoE-KO mice and endothelial cells (EC) were used for the study. Aortic atherogenic lesions in PYK2-KO/ApoE-KO mice were markedly decreased (55% versus ApoE-KO) after 8 weeks of a Western diet. Aortic PYK2 was activated as early as 7 days after the Western diet, when inflammatory cells were not yet activated. Addition of the proatherogenic oxidized phospholipid lysophosphatidylcholine caused activation of endothelial PYK2. Lysophosphatidylcholine-activated PYK2 induced NADPH oxidase-mediated ROS generation and ROS-mediated synthesis of tumor necrosis factor-α (TNFα), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1), and p21Cip1/Ets-1. Neutralizing anti-TNFα antibody or knockdown of p21Cip1/Ets-1 system blocked the induction of VCAM-1 and MCP-1. PYK2 deficiency abolished these ROS-mediated proinflammatory reactions. Further analysis revealed that PYK2/ROS-mediated p21Cip1/Ets-1 activation upregulated the transcription of the MCP-1 gene in collaboration with p300 transcription coactivator. CONCLUSIONS: PYK2 is a key tyrosine kinase activated by high cholesterol exposure, which causes ROS-mediated TNFα release and induces TNFα-dependent expression of proinflammatory molecules via the p21Cip1/Ets-1/p300 transcription system.

Stage-specific role of endogenous Smad2 activation in cardiomyogenesis of embryonic stem cells.

The role of Smads and their specific ligands during cardiomyogenesis in ES cells was examined. Smad2 was activated bimodally in the early and late phases of cardiac differentiation, whereas Smad1 was activated after the middle phase. Nodal and Cripto were expressed in the early stage and then downregulated, whereas transforming growth factor-beta and activin were expressed only in the late phase. Suppression of early Smad2 activation by SB-431542 produced complete inhibition of endodermal and mesodermal induction but augmented neuroectodermal differentiation, followed by poor cardiomyogenesis, whereas inhibition during the late phase alone promoted cardiomyogenesis. Inhibitory effect of Smad2 on cardiomyogenesis in the late phase was mainly mediated by transforming growth factor-beta, and inhibition of transforming growth factor-beta-mediated Smad2 activation resulted in a greater replicative potential in differentiated cardiac myocytes and enhanced differentiation of nonmyocytes into cardiac myocytes. Thus, endogenous Smad2 activation is indispensable for endodermal and mesodermal induction in the early phase. In the late phase, endogenous transforming growth factor-beta negatively regulates cardiomyogenesis through Smad2 activation by modulating proliferation and differentiation of cardiac myocytes.

Skeletal muscle-derived progenitors capable of differentiating into cardiomyocytes proliferate through myostatin-independent TGF-beta family signaling.

The existence of skeletal muscle-derived stem cells (MDSCs) has been suggested in mammals; however, the signaling pathways controlling MDSC proliferation remain largely unknown. Here we report the isolation of myosphere-derived progenitor cells (MDPCs) that can give rise to beating cardiomyocytes from adult skeletal muscle. We identified that follistatin, an antagonist of TGF-beta family members, was predominantly expressed in MDPCs, whereas myostatin was mainly expressed in myogenic cells and mature skeletal muscle. Although follistatin enhanced the replicative growth of MDPCs through Smad2/3 inactivation and cell cycle progression, disruption of myostatin did not increase the MDPC proliferation. By contrast, inhibition of activin A (ActA) or growth differentiation factor 11 (GDF11) signaling dramatically increased MDPC proliferation via down-regulation of p21 and increases in the levels of cdk2/4 and cyclin D1. Thus, follistatin may be an effective progenitor-enhancing agent neutralizing ActA and GDF11 signaling to regulate the growth of MDPCs in skeletal muscle.

Local overexpression of toll-like receptors at the vessel wall induces atherosclerotic lesion formation: synergism of TLR2 and TLR4.

OBJECTIVE: Atherosclerosis is now considered as a chronic inflammatory disease, and inflammation is closely related to immune systems, which consist of innate-immunity and adaptive-immunity. Recently, toll-like receptors (TLRs) have been identified as key components of innate-immunity. We examined the role of local expressions of TLRs at the vessel wall in atherosclerosis. METHODS AND RESULTS: We transfected cDNA encoding human TLR2 and TLR4 into the carotid arterial vessel wall of rabbits fed high-cholesterol diets with the use of HVJ-liposome. The rabbits were transfected with (1) pCMV-beta-gal, (2) empty vector, (3) TLR2, (4) TLR4, (5) TLR2+4. X-gal staining and immunohistochemical analysis showed that the transfected plasmids were mainly expressed in the media. Neither TLR2 nor TLR4 transfection induced significant augmentation of atherosclerosis. Transfection of TLR2- and TLR4-containing HVJ synergistically accelerated atherosclerosis and increased expressions of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and MCP-1. Moreover, transfection of TLR2 and TLR4 resulted in synergistic activation of NF-kappaB at the vessel wall in vivo, and in vascular smooth muscle cells in vitro. CONCLUSIONS: Expressions of both TLR2 and TLR4 at the vessel wall synergistically accelerated atherosclerosis. The present study revealed the role of TLRs expressed locally at the vessel wall in the early stage of atherosclerosis.