Anh Cao

Conventional B2 B Cell Depletion Ameliorates whereas Its Adoptive Transfer Aggravates Atherosclerosis

Atherosclerosis is a chronic inflammatory arterial disease characterized by focal accumulation of lipid and inflammatory cells. It is the number one cause of deaths in the Western world because of its complications of heart attacks and strokes. Statins are effective in only approximately one third of patients, underscoring the urgent need for additional therapies. B cells that accumulate in atherosclerotic lesions and the aortic adventitia of humans and mice are considered to protect against atherosclerosis development. Unexpectedly, we found that selective B cell depletion in apolipoprotein E-deficient (ApoE−/−) mice using a well-characterized mAb to mouse CD20 reduced atherosclerosis development and progression without affecting the hyperlipidemia imposed by a high-fat diet. Adoptive transfer of 5 × 106 or 5 × 107 conventional B2 B cells but not 5 × 106 B1 B cells to a lymphocyte-deficient ApoE−/− Rag-2−/− common cytokine receptor γ-chain–deficient mouse that was fed a high-fat diet augmented atherosclerosis by 72%. Transfer of 5 × 106 B2 B cells to an ApoE−/− mouse deficient only in B cells aggravated atherosclerosis by >300%. Our findings provide compelling evidence for the hitherto unrecognized proatherogenic role of conventional B2 cells. The data indicate that B2 cells can potently promote atherosclerosis development entirely on their own in the total absence of all other lymphocyte populations. Additionally, these B2 cells can also significantly augment atherosclerosis development in the presence of T cells and all other lymphocyte populations. Our findings raise the prospect of B cell depletion as a therapeutic approach to inhibit atherosclerosis development and progression in humans.

Cytotoxic and Proinflammatory CD8+ T Lymphocytes Promote Development of Vulnerable Atherosclerotic Plaques in ApoE-Deficient Mice

Background— Heart attacks and strokes, leading causes of deaths globally, arise from thrombotic occlusion of ruptured vulnerable atherosclerotic plaques characterized by abundant apoptosis, large necrotic cores derived from inefficient apoptotic cell clearance, thin fibrous caps, and focal inflammation. The genesis of apoptosis and necrotic cores in these vulnerable atherosclerotic plaques remains unknown. Cytotoxic CD8+ T lymphocytes represent up to 50% of leukocytes in advanced human plaques and dominate early immune responses in mouse lesions, yet their role in atherosclerosis also remains unresolved. Methods and Results— CD8+ T-lymphocyte depletion by CD8&agr; or CD8&bgr; monoclonal antibody in apolipoprotein E-deficient mice fed a high-fat diet ameliorated atherosclerosis by reducing lipid and macrophage accumulation, apoptosis, necrotic cores, and monocyte chemoattractant protein 1, interleukin 1&bgr;, interferon &ggr;, and vascular cell adhesion molecule 1. Transfer of CD8+ T cells into lymphocyte-deficient, apolipoprotein E-deficient mice partially reconstituted CD8+ T cells in lymphoid compartments and was associated with CD8+ T-cell infiltration in lesions, increased lipid and macrophage accumulation, apoptotic cells, necrotic cores, and interleukin 1&bgr; in atherosclerotic lesions. Transfer of CD8+ T cells deficient in perforin, granzyme B, or tumor necrosis factor &agr; but not interferon &ggr; failed to increase atherosclerotic lesions despite partial reconstitution in the lymphoid system and the presence in atherosclerotic lesions. Macrophages, smooth muscle cells, and endothelial cells were identified as apoptotic targets. Conclusions— We conclude that CD8+ T lymphocytes promote the development of vulnerable atherosclerotic plaques by perforin- and granzyme B–mediated apoptosis of macrophages, smooth muscle cells, and endothelial cells that, in turn, leads to necrotic core formation and further augments inflammation by tumor necrosis factor &agr; secretion.

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation.

Background New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated. Methods Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angelis salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined. Results We now demonstrate that Angelis salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angelis salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angelis salt were mimicked by BNP. We also demonstrate that the effects of Angelis salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP). Conclusions Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.

Annexin-1 peptide Anx-12–26 protects adult rat cardiac myocytes from cellular injury induced by simulated ischaemia

1 The anti‐inflammatory properties of annexin‐1 peptides have been largely ascribed to their powerful antineutrophil actions in vivo. We have recently reported that the N‐terminal fragment of annexin‐1, Anx‐12–26, preserves contractile function of cardiac muscle in vitro. The aim of the present study was to determine if Anx‐12–26 elicits protective actions specifically on the cardiac myocyte (in the absence of neutrophils), using a model of metabolic inhibition to simulate ischaemia. 2 Metabolic inhibition of cardiac myocytes (4 h incubation at 37°C in HEPES‐containing buffer supplemented with 2‐deoxy‐D‐glucose, D,L‐lactic acid and pH adjusted to 6.5) followed by 2.5 h recovery in normal medium markedly increased creatine kinase (CK) and lactate dehydrogenase (LDH) levels by 179±39 and 26±7 IU L−1 (both n=40, P<0.001), respectively. However, cellular injury was significantly decreased when Anx‐12–26 (0.3 μM) was present during metabolic inhibition, CK by 74±10% and LDH by 71±6% (both n=31, P<0.001), respectively. 3 Boc 2 (10 μM), a nonselective formyl peptide receptor antagonist, present during metabolic inhibition, abolished the cardioprotective effect of Anx‐12–26. 4 Addition of chelerythrine (10 μM), 5‐hydroxydecanoate (500 μM) or SB202190 (1 μM) during metabolic inhibition also abolished Anx‐12–26‐induced cardioprotection. 5 Cellular injury induced by metabolic inhibition was also largely prevented when myocytes were incubated with Anx‐12–26 for 5 min with 10 min recovery prior to the insult, or when Anx‐12–26 was present only during the recovery period following drug‐free metabolic inhibition. 6 In conclusion, the annexin‐1 peptide Anx‐12–26 potently prevents cardiac myocyte injury induced by metabolic inhibition, an action that was dependent at least in part on the activation of the formyl peptide receptor family of G‐protein‐coupled receptors, protein kinase C, p38 mitogen‐activated protein kinase and ATP‐sensitive potassium channels.

Phosphatidylserine Liposomes Mimic Apoptotic Cells to Attenuate Atherosclerosis by Expanding Polyreactive IgM Producing B1a lymphocytes

AIMS To investigate whether activation of atheroprotective peritoneal B1a cells by apoptotic cells or phosphatidylserine liposomes (PSLs) can enhance their protective actions during atherosclerosis development. METHODS AND RESULTS Male apolipoprotein E-knockout (ApoE-/-) mice were treated with apoptotic cells or PSLs at the beginning of 8-week high-fat diet. Intraperitoneally administered apoptotic cells attenuated atherosclerosis in hypercholesterolemic ApoE-/- mice by 53% and macrophage accumulation by 52%, effects mimicked by administering PSLs and abolished by B1a cell depletion by splenectomy. These effects were associated with reduced lesion CD4+ and CD8+ T cells, mRNAs of MCP-1, VCAM-1, TNF-α, IL-1β, IL-12, and IL-18 while anti-inflammatory TGF-β mRNA levels doubled. Apoptotic cells or PSLs increased B1a lymphocytes including TIM-1+ B1a cells in vivo and in vitro while other lymphocyte populations were unaffected. Total plasma IgM, anti-leucocyte, anti-CD3, anti-CD4, and anti-oxLDL IgM were elevated. IgM in atherosclerotic lesions was also elevated and this was associated with reduced lesion MDA-LDL (oxLDL), apoptotic cells and necrotic core size. These effects of activating B1a cells could be attributed to B1a-derived polyreactive IgM deposited in lesions that reduce inflammatory cytokines by lowering lesion ox-LDL via anti-oxLDL IgM, T-cells via anti-leucocyte, anti-CD3, and anti-CD4 IgM, apoptotic cells and necrotic core size via IgM binding to apoptotic cells and enhancing phagocytosis, which also elevates anti-inflammatory cytokines. CONCLUSION Targeting B1a cell activation by PSLs may be a potentially potent therapeutic strategy to attenuate atherosclerosis and reduce the incidence of atherosclerosis-dependent myocardial infarction and stroke.

Atrial natriuretic peptide prevents diabetes-induced endothelial dysfunction.

Atrial natriuretic peptide (ANP) exerts beneficial effects on the cardiovascular system in part by exerting antioxidant activity. Given that oxidant stress is a key cause of endothelial dysfunction in diabetes, we investigated whether ANP improves endothelial function in rats with diabetes. Rats were injected with streptozotocin (55 mg/kg iv) to induce type 1 diabetes or the citrate vehicle as controls (n=12). After 4 weeks the diabetic rats were treated with ANP (10 pmol/kg/min sc, n=12) or the antioxidant tempol (1.5 mmol/kg/day sc, n=11), both by osmotic minipump, ramipril (1 mg/kg per day in the drinking water) or remained untreated (n=11). After a further 4 weeks, anaesthetised rats were killed by exsanguination and the thoracic aortae collected for examination of vascular activity and measurement of superoxide generation. Diabetic rats showed elevated plasma glucose concentration (45+/-3 mM) compared to controls (10+/-1 mM) and this was not affected by ANP (43+/-3 mM), ramipril (41+/-2 mM) or tempol (43+/-2 mM). Endothelium-dependent relaxation ex vivo in response to acetylcholine was impaired in diabetic rats (Rmax=66+/-4%) compared to control rats (Rmax=94+/-1%) but treatment with ANP (Rmax=80+/-4%), ramipril (Rmax=88+/-2%) or tempol (Rmax=81+/-5%) significantly improved those responses. Relaxant responses to the endothelium-independent vasodilator sodium nitroprusside were enhanced by treatment of diabetic rats with ANP or ramipril and their combination; but not by tempol. Superoxide generation was significantly elevated in aorta from untreated diabetic rats (649+/-146% of control). In diabetic rats, superoxide generation was significantly attenuated by ANP (to 229+/-78%) or tempol (to 186+/-64%). This study demonstrates that ANP improves vascular oxidant stress in concert with endothelial function, independent of any effect on plasma glucose levels. These studies may lead to new therapies, based on natriuretic peptide and/or antioxidant approaches, for ameliorating the vascular complications of diabetes.

Sodium nitroprusside protects adult rat cardiac myocytes from cellular injury induced by simulated ischemia: Role for a non-cGMP-dependent mechanism of nitric oxide protection

The cardioprotective actions of nitric oxide (NO) have largely been attributed to cGMP. NO may, however, elicit some biological actions independently of cGMP. We tested the hypothesis that the NO donor sodium nitroprusside specifically protects isolated cardiomyocytes from injury at least in part independently of its ability to elevate cGMP by using metabolic inhibition to simulate ischemia. Metabolic inhibition-induced injury of adult rat cardiomyocytes (increased activity of lactate dehydrogenase and creatine kinase) was significantly reduced by sodium nitroprusside by at least 30% at all concentrations studied (0.3-100 μM). Sodium nitroprusside (1 μM) increased cardiomyocyte cGMP content, but neither a stable analogue of cGMP (8-bromo-cGMP) nor a potent cGMP stimulus (atrial natriuretic peptide) mimicked the protective effects of sodium nitroprusside. Moreover, inhibition of soluble guanylyl cyclase failed to inhibit sodium nitroprusside cardiomyocyte protection. Conversely, inhibition of either ATP-sensitive potassium (KATP) channels with glibenclamide (10 μM) or calcium-sensitive potassium (KCa) channels with tetraethylammonium bromide (1 mM) or iberiotoxin (20 nM) markedly attenuated the cardioprotective actions of sodium nitroprusside. In conclusion, sodium nitroprusside protects isolated cardiomyocytes from metabolic inhibition independently of cGMP; rather, inhibition of KCa and KATP channels reverses the sodium nitroprusside actions, thus unmasking another mechanism for NO-mediated protection in cardiomyocytes.

B-cell-specific depletion of tumour necrosis factor alpha inhibits atherosclerosis development and plaque vulnerability to rupture by reducing cell death and inflammation

AIMS B2 lymphocytes promote atherosclerosis development but their mechanisms of action are unknown. Here, we investigated the role of tumour necrosis factor alpha (TNF-α) produced by B2 cells in atherogenesis. METHODS AND RESULTS We found that 50% of TNF-α-producing spleen lymphocytes were B2 cells and ∼20% of spleen and aortic B cells produced TNF-α in hyperlipidemic ApoE(-/-) mice. We generated mixed bone marrow (80% μMT/20% TNF-α(-/-)) chimeric LDLR(-/-) mice where only B cells did not express TNF-α. Atherosclerosis was reduced in chimeric LDLR(-/-) mice with TNF-α-deficient B cells. TNF-α expression in atherosclerotic lesions and in macrophages were also reduced accompanied by fewer apoptotic cells, reduced necrotic cores, and reduced lesion Fas, interleukin-1β and MCP-1 in mice with TNF-α-deficient B cells compared to mice with TNF-α-sufficient B cells. To confirm that the reduced atherosclerosis is attributable to B2 cells, we transferred wild-type and TNF-α-deficient B2 cells into ApoE(-/-) mice deficient in B cells or in lymphocytes. After 8 weeks of high fat diet, we found that atherosclerosis was increased by wild-type but not TNF-α-deficient B2 cells. Lesions of mice with wild-type B2 cells but not TNF-α-deficient B2 cells also had increased apoptotic cells and necrotic cores. Transferred B2 cells were found in lesions of recipient mice, suggesting that TNF-α-producing B2 cells promote atherosclerosis within lesions. CONCLUSION We conclude that TNF-α produced by B2 cells is a key mechanism by which B2 cells promote atherogenesis through augmenting macrophage TNF-α production to induce cell death and inflammation that promote plaque vulnerability.

A CD1d-dependent lipid antagonist to NKT cells ameliorates atherosclerosis in ApoE−/− mice by reducing lesion necrosis and inflammation

AIMS Atherosclerosis-related deaths from heart attacks and strokes remain leading causes of global mortality, despite the use of lipid-lowering statins. Thus, there is an urgent need to develop additional therapies. METHODS AND RESULTS Reports that NKT cells promote atherosclerosis and an NKT cell CD1d-dependent lipid antagonist (DPPE-PEG350, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N[methoxy(polyethyleneglycol)-350]) reduces allergen-induced inflammation led us to investigate its therapeutic potential in preventing the development and progression of experimental atherosclerosis. DPPE-PEG350 was administered to hyperlipidaemic ApoE(-/-) mice with/without established atherosclerosis. Atherosclerosis and immune cells were assessed in the aortic sinus lesions. Lesion expression of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion protein-1 (VCAM-1) responsible for inflammatory immune cell recruitment as well as mRNA expression of IFNγ and its plasma levels were investigated. Necrotic cores and lesion smooth muscle and collagen contents important in plaque stability were determined as were plasma lipid levels. DPPE-PEG350 reduced atherosclerosis development and delayed progression of established atherosclerosis without affecting plasma lipids. CD4 and CD8 T cells and B cells in atherosclerotic lesions were decreased in DPPE-PEG350-treated mice. Lesion MCP-1 and VCAM-1 protein expression and necrotic core size were reduced without affecting lesion smooth muscle and collagen content. IFNγ and lymphocytes were unaffected by the treatment. CONCLUSION The attenuation of progression of established atherosclerosis together with reduced development of atherosclerosis in hyperlipidaemic mice by the NKT antagonist, without affecting NKT cell or other lymphocyte numbers, suggests that targeting lesion inflammation via CD1d-dependent activation of NKT cells using DPPE-PEG350 has a therapeutic potential in treating atherosclerosis.

Insulin replacement limits progression of diabetic cardiomyopathy in the low-dose streptozotocin-induced diabetic rat

Diabetic cardiomyopathy is a major contributor to the increasing burden of heart failure globally. Effective therapies remain elusive, in part due to the incomplete understanding of the mechanisms underlying diabetes-induced myocardial injury. The objective of this study was to assess the direct impact of insulin replacement on left ventricle structure and function in a rat model of diabetes. Male Sprague-Dawley rats were administered streptozotocin (55 mg/kg i.v.) or citrate vehicle and were followed for 8 weeks. A subset of diabetic rats were allocated to insulin replacement (6 IU/day insulin s.c.) for the final 4 weeks of the 8-week time period. Diabetes induced the characteristic systemic complications of diabetes (hyperglycaemia, polyuria, kidney hypertrophy) and was accompanied by marked left ventricle remodelling (cardiomyocyte hypertrophy, left ventricle collagen content) and diastolic dysfunction (transmitral E/A, left ventricle-dP/dt). Importantly, these systemic and cardiac impairments were ameliorated markedly following insulin replacement, and moreover, markers of the diabetic cardiomyopathy phenotype were significantly correlated with the extent of hyperglycaemia. In summary, these data suggest that poor glucose control directly contributes towards the underlying features of experimental diabetic cardiomyopathy, at least in the early stages, and that adequate replacement ameliorates this.