Rui-Hai Zhou

Intervertebral Disc Degeneration: The Role of the Mitochondrial Pathway in Annulus Fibrosus Cell Apoptosis Induced by Overload

Degeneration of the intervertebral disk (IVD) is a major pathological process implicated in low back pain and is a prerequisite to disk herniation. Although mechanical stress is an important modulator of the degeneration, the underlying molecular mechanism remains unclear. The association of human IVD degeneration, assessed by magnetic resonance imaging, with annulus fibrosus cell apoptosis and anti-cytochrome c staining revealed that the activation of the mitochondria-dependent apoptosome was a major event in the degeneration process. Mouse models of IVD degeneration were used to investigate the role of the mechanical stress in this process. The application of mechanical overload (1.3 MPa) for 24 hours induced annulus fibrosus cell apoptosis and led to severe degeneration of the mouse disks. Immunostaining revealed cytochrome c release but not Fas-L generation. The role of the caspase-9-dependent mitochondrial pathway in annulus fibrosus cell apoptosis induced by overload was investigated further with the use of cultured rabbit IVD cells in a stretch device. Mechanical overload (15% area change) induced apoptosis with increased caspase-9 activity and decreased mitochondrial membrane potential. Furthermore, Z-LEHD-FMK, a caspase-9 inhibitor, but not Z-IETD-FMK, a caspase-8 inhibitor, attenuated the overload-induced apoptosis. Our results from human samples, mouse models, and annulus fibrosus culture experiments demonstrate that the mechanical overload-induced IVD degeneration is mediated through the mitochondrial apoptotic pathway in IVD cells.

Mitochondrial Oxidative Stress in Aortic Stiffening With Age: The Role of Smooth Muscle Cell Function

Objective—Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Methods and Results—Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/− but not in wild-type, p47phox−/− and SOD1+/− mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/− versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/− mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/− mice. Smooth muscle &agr;-actin levels were increased with age in SOD2+/− versus wild-type SMCs. Aged SOD2+/− SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-&agr;–induced Jun N-terminal kinase 1 activation. Aged SOD2+/− SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/− SMCs. Conclusion—Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

Stent Implantation Activates Akt in the Vessel Wall: Role of Mechanical Stretch in Vascular Smooth Muscle Cells

Objective—The long-term efficacy of stent implantation is affected by in-stent restenosis (ISR). Multiple factors can contribute to ISR, and the underlying mechanism remains elusive. We investigated the possible role of mechanical stretch and the associated molecular signaling in ISR. Methods and Results—Stent implantation in rat abdominal aortas induced neointima formation. Immunohistochemical studies revealed the activation of Akt in the media and neointima of the stented vessels. Western blotting showed increased phosphorylation of Akt at both Thr308 and Ser473 and phosphorylation of GSK-3&bgr; in the stented vessels. A stretch device applying static equibiaxial stretch on cultured vascular smooth muscle cells was used to delineate the molecular mechanism underlying the stretch activation of Akt. Static mechanical stretch induced the sustained activation of Akt and its upstream phosphoinositide 3-kinase (PI3K) and the phosphorylation of GSK-3&bgr;, its downstream effector in vascular smooth muscle cells. LY294002, a PI3K inhibitor, and N-acetylcysteine, a scavenger of reactive oxygen species, inhibited the stretch activation of Akt. Furthermore, N-acetylcysteine and wortmannin, another PI3K inhibitor, reduced the neointima formation after stent implantation. Conclusions—Mechanical stretch of the vascular wall during stent deployment may contribute to ISR by activating the Akt pathway.

Vascular Endothelial Growth Factor Activation of Sterol Regulatory Element Binding Protein: A Potential Role in Angiogenesis

By stimulating the migration and proliferation of endothelial cells (ECs), vascular endothelial growth factor (VEGF) is a potent angiogenic factor. However, the molecular mechanism involved in the VEGF-induced angiogenesis remains elusive. We hypothesized that sterol regulatory element binding proteins (SREBPs), transcription factors governing cellular lipid homeostasis, play an important role in regulating angiogenesis in response to VEGF. VEGF activated SREBP1 and SREBP2 in ECs, as demonstrated by the increased SREBPs, their cleavage products, and the upregulation of the targeted genes. VEGF-induced SREBP activation depended on SREBP cleavage-activating protein (SCAP), because knocking down SCAP by RNA interference (RNAi) inhibited SREBP activation in response to VEGF. SREBP activation was also blocked by 25-hydroxycholesterol (25-HC). To verify the functional implication of SREBPs in VEGF-induced angiogenesis, we tested the role of SREBPs in EC migration and proliferation. SCAP RNAi or 25-HC inhibited VEGF-induced pseudopodia extension and migration of ECs. Both treatments inhibited VEGF-induced EC proliferation, with cell growth arrested at the G0/G1 phase and a concomitant decrease of the S phase. Blocking the PI3K-Akt pathway inhibited the VEGF-activated SREBPs, demonstrating that PI3K-Akt regulates SREBPs. Consistent with our in vitro data, SREBP1 was detected in newly developed microvasculatures in a rabbit skin partial-thickness wound-healing model. SREBP inhibition also markedly suppressed VEGF-induced angiogenesis in chick embryos. In summary, this study identifies SREBPs as the key molecules in regulating angiogenesis in response to VEGF.

Vascular Smooth Muscle Overexpression of G Protein–Coupled Receptor Kinase 5 Elevates Blood Pressure, Which Segregates With Sex and Is Dependent on Gi-Mediated Signaling

Background—Essential hypertension involves an increase in sympathetic nervous system activity and an associated decrease in &bgr;-adrenergic receptor (AR)–mediated dilation. In addition, increased levels of G protein–coupled receptor (GPCR) kinases (GRKs), which regulate GPCR signaling, are associated with increased blood pressure (BP). Methods and Results—We generated transgenic mice with ≈2-fold vascular smooth muscle (VSM)–specific overexpression of GRK5 to recapitulate a selective aspect of hypertension and understand the impact on GPCR regulation of BP. VSM-GRK5 mice were hypertensive, with a 25% to 35% increase in BP, whereas there was no concomitant cardiac or VSM hypertrophy. BP elevations were segregated with sex, with male mice having higher levels than female mice, and ovariectomy did not alter this phenotype. BP was restored to control values with pertussis toxin Gi-signaling inhibition or chronic &bgr;1AR inhibition after 7 days of CGP20712A, whereas the &bgr;2AR antagonist ICI 118,551 was ineffective. &agr;1AR response was not altered, nor was &bgr;AR-mediated dilation in male blood vessels, whereas norepinephrine sensitivity was increased. In contrast, female VSM-GRK5 blood vessels have diminished &bgr;AR-mediated dilation and enhanced sensitivity to angiotensin II (Ang II). Conclusions—Our data suggest that in both male and female mice, VSM-specific overexpression of GRK5 elevates BP mediated by Gi and, at least in part, by &bgr;1AR in males and Ang II receptors in females. Understanding mechanisms underlying an increase in VSM-GRK5 may have a profound influence on the use and development of antihypertensive therapeutics.

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances α1D-adrenergic receptor constriction

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

Impaired neoangiogenesis in β2-adrenoceptor gene-deficient mice: restoration by intravascular human β2-adrenoceptor gene transfer and role of NFκB and CREB transcription factors

There is much evidence supporting the role of β2‐adrenoceptors (β2AR) in angiogenesis but the mechanisms underlying their effects have not been elucidated. Hence, we studied post‐ischaemic angiogenesis in the hindlimb (HL) of β2AR knock‐out mice (β2AR−/−) in vivo and explored possible molecular mechanisms in vitro.

Proteomic analysis of aorta and protective effects of grape seed procyanidin B2 in db/db mice reveal a critical role of milk fat globule epidermal growth factor-8 in diabetic arterial damage.

Background Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM. Methods and Results GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2. Conclusion Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.

Proteomic analysis of kidney and protective effects of grape seed procyanidin B2 in db/db mice indicate MFG-E8 as a key molecule in the development of diabetic nephropathy

Diabetic nephropathy, as a severe microvascular complication of diabetic mellitus, has become the leading cause of end-stage renal diseases. However, no effective therapeutic strategy has been developed to prevent renal damage progression to end stage renal disease. Hence, the present study evaluated the protective effects of grape seed procyanidin B2 (GSPB2) and explored its molecular targets underlying diabetic nephropathy by a comprehensive quantitative proteomic analysis in db/db mice. Here, we found that oral administration of GSPB2 significantly attenuated the renal dysfunction and pathological changes in db/db mice. Proteome analysis by isobaric tags for relative and absolute quantification (iTRAQ) identified 53 down-regulated and 60 up-regulated proteins after treatment with GSPB2 in db/db mice. Western blot analysis confirmed that milk fat globule EGF-8 (MFG-E8) was significantly up-regulated in diabetic kidney. MFG-E8 silencing by transfection of MFG-E8 shRNA improved renal histological lesions by inhibiting phosphorylation of extracellular signal-regulated kinase1/2 (ERK1⁄2), Akt and glycogen synthase kinase-3beta (GSK-3β) in kidneys of db/db mice. In contrast, over-expression of MFG-E8 by injection of recombinant MFG-E8 resulted in the opposite effects. GSPB2 treatment significantly decreased protein levels of MFG-E8, phospho-ERK1/2, phospho-Akt, and phospho-GSK-3β in the kidneys of db/db mice. These findings yield insights into the pathogenesis of diabetic nephropathy, revealing MFG-E8 as a new therapeutic target and indicating GSPB2 as a prospective therapy by down-regulation of MFG-E8, along with ERK1/2, Akt and GSK-3β signaling pathway.

Activation of sterol regulatory element‐binding proteins (SREBPs) is critical in IL‐8‐induced angiogenesis

Angiogenesis is essential in many physiological and pathological processes and can be stimulated by many different fators. To better understand and to manipulate this process more effectively, it would be beneficial to identify molecules common to the signaling pathways stimulated by different classes of angiogenic factors. Sterol regulatory element‐binding proteins (SREBPs) are involved in the metabolism of cholesterol and fatty acids, molecules that are critical in membrane biology, and hence, many of the processes involved in angiogenesis. Here, we show that angiogenic factors of different families, such as basic fibroblast growth factor, thrombin, and interleukin (IL)‐8, stimulate SREBP activation, whereas nonangiogenic factors, such as transforming growth factor‐β1, do not. We focused our detailed studies on IL‐8 in vitro and in vivo, as this chemokine is also involved in inflammation and hence, has the potential to be critical in inflammation‐induced angiogenesis, a process common to many diseases. Using human microvascular endothelial cells, a rabbit skin wound‐healing model, and the chorioallantoic membrane assay, we show that IL‐8 stimulates the activation of SREBP‐1 and ‐2, and this activation is specific and receptor‐mediated. SREBP activation leads to activation of RhoA through 3‐hydroxy‐3‐methylglutaryl CoA reductase. RhoA is a small guanosinetriphosphatase, important in cytoskeletal functions, which in turn, are critical in many of the cellular processes needed for angiogenesis. Given that diverse, angiogenic factors use different cell‐surface receptors, identification of this common step in the signal‐transduction pathway provides the opportunity for novel approaches for prevention and treatment of diseases involving abnormal angiogenesis.