Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

mTOR Hyperactivation by Ablation of Tuberous Sclerosis Complex 2 in the Mouse Heart Induces Cardiac Dysfunction with the Increased Number of Small Mitochondria Mediated through the Down-Regulation of Autophagy.

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts.

Unique remodeling processes after vascular injury in intracranial arteries: analysis using a novel mouse model.

The effectiveness of angioplasty and stenting in intracranial atherosclerotic diseases is controversial due to high rates of delayed restenosis and hemorrhage compared with extracranial arteries. However, the mechanisms underlying these differences are still unclear, because their pathophysiology is yet to be examined. To address this issue, we established a novel vascular injury model in the intracranial internal carotid arteries (IICAs) in mice, and analyzed the remodeling process in comparison to that of the femoral arteries (FAs). In IICAs, neointimal hyperplasia was observed from day 14 and grew until day 56. Although smooth muscle cells (SMCs) emerged in the neointima from day 28, SMCs in the injured media were continuously lost with eventual extinction of the media. Re-endothelialization was started from day 7 and completed on day 28. Accumulation of macrophages was continued in the adventitia until day 56. Compared with FAs, the following points are unique in IICAs: (1) delayed continuous formation of neointima; (2) accumulation of macrophages in the media on day 14; (3) continuous loss of SMCs in the media followed by extinction of the media itself; and (4) continuously growing adventitia. These pathophysiologic differences might be associated with unfavorable outcomes in percutaneous transluminal angioplasty and stenting in intracranial arteries.

The ATP-binding cassette transporter ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

OBJECTIVE: ATP-binding cassette transporter subfamily G member 2 (ABCG2), expressed in microvascular endothelial cells in the heart, has been suggested to regulate several tissue defense mechanisms. This study was performed to elucidate its role in pressure overload-induced cardiac hypertrophy. METHODS AND RESULTS: Pressure overload was induced in 8- to 12-week-old wild-type and Abcg2-/- mice by transverse aortic constriction (TAC). Abcg2-/- mice showed exaggerated cardiac hypertrophy and ventricular remodeling after TAC compared with wild-type mice. In the early phase after TAC, functional impairment in angiogenesis and antioxidant response in myocardium was found in Abcg2-/- mice. In vitro experiments demonstrated that ABCG2 regulates transport of glutathione, an important endogenous antioxidant, from microvascular endothelial cells. Besides, glutathione transported from microvascular endothelial cells in ABCG2-dependent manner ameliorated oxidative stress-induced cardiomyocyte hypertrophy. In vivo, glutathione levels in plasma and the heart were increased in wild-type mice but not in Abcg2-/- mice after TAC. Treatment with the superoxide dismutase mimetic ameliorated cardiac hypertrophy in Abcg2-/- mice after TAC to the same extent as that in wild-type mice, although cardiac dysfunction with impaired angiogenesis was observed in Abcg2-/- mice. CONCLUSION: ABCG2 protects against pressure overload-induced cardiac hypertrophy and heart failure by promoting angiogenesis and antioxidant response.

The ATP-binding cassette transporter BCRP1/ABCG2 plays a pivotal role in cardiac repair after myocardial infarction via modulation of microvascular endothelial cell survival and function.

OBJECTIVE: To clarify the impact of breast cancer resistance protein 1 (BCRP1)/ATP-binding cassette transporter subfamily G member 2 (ABCG2) expression on cardiac repair after myocardial infarction (MI). METHODS AND RESULTS: The ATP-binding cassette transporter BCRP1/ABCG2 is expressed in various organs, including the heart, and may regulate several tissue defense mechanisms. BCRP1/ABCG2 was mainly expressed in endothelial cells of microvessels in the heart. MI was induced in 8- to 12-week-old wild-type (WT) and Bcrp1/Abcg2 knockout (KO) mice by ligating the left anterior descending artery. At 28 days after MI, the survival rate was significantly lower in KO mice than in WT mice because of cardiac rupture. Echocardiographic, hemodynamic, and histological assessments showed that ventricular remodeling was more deteriorated in KO than in WT mice. Capillary, myofibroblast, and macrophage densities in the peri-infarction area at 5 days after MI were significantly reduced in KO compared with WT mice. In vitro experiments demonstrated that inhibition of BCRP1/ABCG2 resulted in accumulation of intracellular protoporphyrin IX and impaired survival of microvascular endothelial cells under oxidative stress. Moreover, BCRP1/ABCG2 inhibition impaired migration and tube formation of endothelial cells. CONCLUSIONS: BCRP1/ABCG2 plays a pivotal role in cardiac repair after MI via modulation of microvascular endothelial cell survival and function.

Endovascular injury induces rapid phenotypic changes in perivascular adipose tissue.

OBJECTIVE: Accumulating evidence suggests that adipose tissue not only stores energy but also secretes various bioactive substances called adipocytokines. Periadventitial fat is distributed ubiquitously around arteries throughout the body. It was reported that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular diseases accelerated by obesity. We investigated the effect of endovascular injury on the phenotype of perivascular fat. METHODS AND RESULTS: Endovascular injury significantly upregulated proinflammatory adipocytokines and downregulated adiponectin within periadventitial fat tissue in models of mouse femoral artery wire injury and rat iliac artery balloon injury. Genetic disruption of tumor necrosis factor (TNF)-alpha attenuated upregulation of proinflammatory adipocytokine expression, with reduced neointimal hyperplasia after vascular injury. Local delivery of TNF-alpha to the periadventitial area enhanced inflammatory adipocytokine expression, which was associated with augmented neointimal hyperplasia in TNF-alpha-deficient mice. Conditioned medium from a coculture of 3T3-L1 and RAW264 cells stimulated vascular smooth muscle cell proliferation. An anti-TNF-alpha neutralizing antibody in the coculture abrogated the stimulating effect of the conditioned medium. CONCLUSIONS: Our findings indicate that endovascular injury induces rapid and marked changes in perivascular adipose tissue, mainly mediated by TNF-alpha. It is suggested that the phenotypic changes in perivascular adipose tissue may have a role in the pathogenesis of neointimal hyperplasia after angioplasty.

Periadventitial adipose tissue plays a critical role in vascular remodeling.

RATIONALE: Obesity is associated with a high incidence of cardiovascular complications. However, the molecular link between obesity and vascular disease is not fully understood. Most previous studies have focused on the association between cardiovascular disease and accumulation of visceral fat. Periadventitial fat is distributed ubiquitously around arteries throughout the body. OBJECTIVE: Here, we investigated the impact of obesity on inflammation in the periadventitial adipose tissue and on lesion formation after vascular injury. METHODS AND RESULTS: High-fat, high-sucrose feeding induced inflammatory changes and decreased adiponectin expression in the periadventitial adipose tissue, which was associated with enhanced neointima formation after endovascular injury. Removal of periadventitial fat markedly enhanced neointima formation after injury, which was attenuated by transplantation of subcutaneous adipose tissue from mice fed on regular chow. Adiponectin-deficient mice showed markedly enhanced lesion formation, which was reversed by local delivery, but not systemic administration, of recombinant adiponectin to the periadventitial area. The conditioned medium from subcutaneous fat attenuated increased cell number of smooth muscle cells in response to platelet derived growth factor-BB. CONCLUSIONS: Our findings suggest that periadventitial fat may protect against neointimal formation after angioplasty under physiological conditions and that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular disease accelerated by obesity.

Treatment with recombinant placental growth factor (PlGF) enhances both angiogenesis and arteriogenesis and improves survival after myocardial infarction.

BACKGROUND: Placental growth factor (PlGF), a homolog of vascular endothelial growth factor, is reported to stimulate angiogenesis and arteriogenesis in pathological conditions. It was recently demonstrated that PlGF is rapidly produced in myocardial tissue during acute myocardial infarction (MI). However, the effects of exogenous PlGF administration on the healing process after MI are not fully understood. The purpose of the present study was to examine whether PlGF treatment has therapeutic potential in MI. METHODS AND RESULTS: Recombinant human PlGF (rhPlGF: 10 microg) was administered continuously for 3 days in a mouse model of acute MI. rhPlGF treatment significantly improved survival rate after MI and preserved cardiac function relative to control mice. The numbers of CD31-positive cells and alpha-smooth muscle actin-positive vessels in the infarct area were significantly increased in the rhPlGF group. Endothelial progenitor cells (Flk-1(+)Sca-1(+) cells) were mobilized by rhPlGF into the peripheral circulation. Furthermore, rhPlGF promoted the recruitment of GFP-labeled bone marrow cells to the infarct area, but only a few of those migrating cells differentiated into endothelial cells. CONCLUSIONS: Exogenous PlGF plays an important role in healing processes by improving cardiac function and stimulating angiogenesis following MI. It can be considered as a new therapeutic molecule.

Epac1 is upregulated during neointima formation and promotes vascular smooth muscle cell migration.

Vascular remodeling after mechanoinjury largely depends on the migration of smooth muscle cells, an initial key step to wound healing. However, the role of the second messenger system, in particular, the cAMP signal, in regulating such remodeling remains controversial. Exchange protein activated by cAMP (Epac) has been identified as a new target molecule of the cAMP signal, which is independent from PKA. We thus examined whether Epac plays a distinct role from PKA in vascular remodeling. To examine the role of Epac and PKA in migration, we used primary culture smooth muscle cells from both the fetal and adult rat aorta. A cAMP analog selective to PKA, 8-(4-parachlorophenylthio)-cAMP (pCPT-cAMP), decreased cell migration, whereas an Epac-selective analog, 8-pCPT-2'-O-Me-cAMP, enhanced migration. Adenovirus-mediated gene transfer of PKA decreased cell migration, whereas that of Epac1 significantly enhanced cell migration. Striking morphological differences were observed between pCPT-cAMP- and 8-pCPT-2'-O-Me-cAMP-treated aortic smooth muscle cells. Furthermore, overexpression of Epac1 enhanced the development of neointimal formation in fetal rat aortic tissues in organ culture. When the mouse femoral artery was injured mechanically in vivo, we found that the expression of Epac1 was upregulated in vascular smooth muscle cells, whereas that of PKA was downregulated with the progress of neointimal thickening. Our findings suggest that Epac1, in opposition to PKA, increases vascular smooth muscle cell migration. Epac may thus play an important role in advancing vascular remodeling and restenosis upon vascular injury.

Inflammatory response to acute myocardial infarction augments neointimal hyperplasia after vascular injury in a remote artery.

OBJECTIVE: Percutaneous coronary intervention (PCI) is currently the most widely accepted treatment for acute myocardial infarction (AMI). It remains unclear, however, whether post-AMI conditions might exacerbate neointimal hyperplasia and restenosis following PCI. Given that both a medial smooth muscle cell lineage and a bone marrow (BM)-derived hematopoietic stem cell lineage are now thought to contribute to neointima formation, the primary aims of the present study were to determine whether AMI augments neointimal hyperplasia at sites of arterial injury, and whether BM-derived cells contribute to that process. METHODS AND RESULTS: We simultaneously generated models of AMI and arterial injury in the same mice, some of which had received BM transplantation. We found that AMI augments neointimal hyperplasia at sites of femoral artery injury by approximately 35% (P<0.05), but that while BM-derived cells contributed to neointimal hyperplasia, they did not contribute to the AMI-related augmentation. Expression of interleukin (IL)-6 mRNA was approximately 7-fold higher in the neointimas of mice subjected to both AMI and arterial injury than in those of mice subjected to arterial injury alone. In addition, we observed increased synthesis of tumor necrosis factor (TNF)-alpha within infarcted hearts and TNF-alpha receptor type 1 (TNFR1) within injured arteries. Chronic treatment with pentoxifylline, which mainly inhibits TNF-alpha synthesis, reduced levels of circulating TNF-alpha and attenuated neointimal hyperplasia after AMI. CONCLUSIONS: Conditions after AMI could exacerbate postangioplasty restenosis, not by increasing mobilization of BM-derived cells, but by stimulating signaling via TNF-alpha, TNFR1 and IL-6.

Cardiac expression of placental growth factor predicts the improvement of chronic phase left ventricular function in patients with acute myocardial infarction.

OBJECTIVES: Our aim was to investigate cardiac expression of placental growth factor (PlGF) and its clinical significance in patients with acute myocardial infarction (AMI). BACKGROUND: Placental growth factor is known to stimulate wound healing by activating mononuclear cells and inducing angiogenesis. The clinical significance of PlGF in AMI is not yet known. METHODS: Fifty-five AMI patients and 43 control subjects participated in the study. Peripheral blood sampling was performed on days 1, 3, and 7 after AMI. Blood was also sampled from the coronary artery (CAos) and the coronary sinus (CS), before and after acute coronary recanalization. Cardiac expression of PlGF was analyzed in a mouse AMI model. RESULTS: In AMI patients, peripheral plasma PlGF levels on day 3 were significantly higher than in control subjects. Plasma PlGF levels just after recanalization were significantly higher in the CS than the CAos, which indicates cardiac production and release of PlGF. Peripheral plasma levels of PlGF on day 3 were negatively correlated with the acute phase left ventricular ejection fraction (LVEF), positively correlated with both acute phase peak peripheral monocyte counts and chronic phase changes in LVEF. Placental growth factor messenger ribonucleic acid expression was 26.6-fold greater in a mouse AMI model than in sham-operated mice, and PlGF was expressed mainly in endothelial cells within the infarct region. CONCLUSIONS: Placental growth factor is rapidly produced in infarct myocardium, especially by endothelial cells during the acute phase of myocardial infarction. Placental growth factor might be over-expressed to compensate the acute ischemic damage, and appears to then act to improve LVEF during the chronic phase.

Inhibitory effect of efonidipine on aldosterone synthesis and secretion in human adrenocarcinoma (H295R) cells.

Targeting aldosterone synthesis and/or release represents a potentially useful approach to the prevention of cardiovascular disease. Aldosterone production is stimulated by angiotensin II (Ang II) or extracellular K+ and is mediated mainly by Ca2+ influx into adrenal glomerulosa cells through T-type calcium channels. We therefore examined the effects of efonidipine, a dual T-type/L-type Ca2+ channel blocker, on aldosterone secretion in the H295R human adrenocarcinoma cell line; 100 nmol/L Ang II and 10 mmol/L K+ respectively increased aldosterone secretion from H295R cells 12-fold and 9-fold over baseline. Efonidipine dose-dependently inhibited both Ang II- and K+-induced aldosterone secretion, and nifedipine, an L-type Ca2+ channel blocker, and mibefradil, a relatively selective T-type channel blocker, similarly inhibited Ang II- and K+-induced aldosterone secretion, but were much less potent than efonidipine. Efonidipine also lowered cortisol secretion most potently among these drugs. Notably, efonidipine and mibefradil also significantly suppressed Ang II- and K+-induced mRNA expression of 11-beta-hydroxylase and aldosterone synthase, which catalyze the final two steps in the aldosterone synthesis, whereas nifedipine reduced only K+-induced enzyme expression. These findings suggest that efonidipine acts via T-type Ca2+ channel blockade to significantly reduce aldosterone secretion, and that this effect is mediated, at least in part, by suppression of 11-beta-hydroxylase and aldosterone synthase expression.

White-coat hypertension contributes to the presence of carotid arteriosclerosis.

It remains unclear whether white-coat hypertension is associated with vascular organ damage (e.g., carotid arteriosclerosis) in the same way sustained hypertension is. We therefore compared the progression of carotid arteriosclerosis among Japanese individuals showing normal blood pressures, sustained hypertension or white-coat hypertension. A total of 30 subjects (mean age, 58 years) with white-coat hypertension, 30 (mean age, 54 years) with untreated sustained hypertension who had no plaque formation in the carotid arteries, and 30 normotensive subjects (mean age, 58 years) were enrolled in this study. The white-coat and sustained hypertensive subjects were matched with respect to their clinical blood pressures, but their ambulatory blood pressures differed. Conversely, white-coat hypertensive and normotensive subjects were matched with respect to ambulatory blood pressures, but their clinical blood pressures differed. Carotid intimal-medial thickness was measured by B-mode ultrasonography, and the cross-sectional area of the common carotid artery was calculated. The three groups were similar with respect to age, sex ratio, height, laboratory data and the incidence of smoking. Body weights and body mass indexes were significantly higher among patients with sustained hypertension than among either normotensive or white-coat hypertensive patients. Intimal-medial thicknesses and carotid cross-sectional areas were similar in patients with white-coat and sustained hypertension and significantly higher than in normotensive subjects. Collectively, these findings suggest that white-coat hypertension contributed to the presence of carotid arteriosclerosis in our subjects in a manner similar to sustained hypertension. Thus, clinical evaluation of white-coat hypertension should be conducted with the potential for target organ damage in mind.