Hua Cai

Publications of Hua Cai

• Role of uncoupled endothelial nitric oxide synthase in abdominal aortic aneurysm formation: treatment with folic acid.

  Authors: Ling Gao, Kin L Siu, Karel Chalupsky, Andrew Nguyen, Peng Chen, Neal L Weintraub, Zorina Galis, Hua Cai

  Hypertension. 11/2011; 59(1):158-66.

  It has been shown that endothelial NO synthase (eNOS) uncoupling occurs in hypertension and atherosclerosis. However, its causal role in vascular pathogenesis has not been characterized previously.It has been shown that endothelial NO synthase (eNOS) uncoupling occurs in hypertension and atherosclerosis. However, its causal role in vascular pathogenesis has not been characterized previously. Here, we challenged eNOS preuncoupled hyperphenylalaninemia (hph)-1 mice (deficient in eNOS cofactor tetrahydrobiopterin biosynthetic enzyme GTPCHI) with angiotensin II (Ang II; 0.7 mg/kg per day, 14 days). Both wild-type and hph-1 groups developed hypertension similarly up to day 6 to 7. Thereafter, ≈14% of Ang II-infused (0.7 mg/kg per day) hph-1 mice (n=72) started to die suddenly of ruptured abdominal aortic aneurysm (AAA). Among the survivors, 65% developed AAA, resulting in a total morbidity rate of 79%. In contrast, none of the Ang II-infused wild-type mice died or developed AAA. Ang II progressively deteriorated eNOS uncoupling in hph-1 mice while augmenting tetrahydrobiopterin and nitric oxide (NO(·)) deficiencies. The abundance of the tetrahydrobiopterin salvage enzyme dihydrofolate reductase in the endothelium was decreased in hph-1 mice and further diminished by Ang II infusion. Intriguingly, restoration of dihydrofolate reductase expression by oral administration of folic acid or overexpression of dihydrofolate reductase completely prevented AAA formation in Ang II-infused hph-1 mice while attenuating progressive uncoupling of eNOS. Folic acid also attenuated vascular remodeling and inflammation characterized by medial elastin breakdown and augmented matrix metalloproteinase 2 activity and activation of matrix metalloproteinase 9, as well as macrophage infiltration. In conclusion, these data innovatively suggest a causal role of eNOS uncoupling/tetrahydrobiopterin deficiency in AAA formation. Therefore, oral folic acid administration, endothelium-targeted dihydrofolate reductase gene therapy, and perhaps other countermeasures directed against eNOS uncoupling could be used as new therapeutics for AAA.

• Repression of P66Shc expression by SIRT1 contributes to the prevention of hyperglycemia-induced endothelial dysfunction.

  Authors: Shuang Zhou, Hou-Zao Chen, Yan-Zhen Wan, Qing-Jun Zhang, Yu-Sheng Wei, Shuai Huang, Jin-Jing Liu, Yun-Biao Lu, Zhu-Qin Zhang, Rui-Feng Yang, Ran Zhang, Hua Cai, De-Pei Liu, Chih-Chuan Liang

  Circulation research. 07/2011; 109(6):639-48.

  Inactivation of the p66Shc adaptor protein confers resistance to oxidative stress and protects mice from aging-associated vascular diseases. However, there is limited information about the negativeInactivation of the p66Shc adaptor protein confers resistance to oxidative stress and protects mice from aging-associated vascular diseases. However, there is limited information about the negative regulating mechanisms of p66Shc expression in the vascular system. Objective: In this study, we investigated the role of SIRT1, a class III histone deacetylase, in the regulation of p66Shc expression and hyperglycemia-induced endothelial dysfunction. Expressions of p66Shc gene transcript and protein were significantly increased by different kinds of class III histone deacetylase (sirtuin) inhibitors in human umbilical vein endothelial cells and 293A cells. Adenoviral overexpression of SIRT1 inhibited high-glucose-induced p66Shc upregulation in human umbilical vein endothelial cells. Knockdown of SIRT1 increased p66Shc expression and also increased the expression levels of plasminogen activator inhibitor-1 expression, but decreased manganese superoxide dismutase expression in high-glucose conditions. However, knockdown of p66Shc significantly reversed the effects of SIRT1 knockdown. In addition, p66Shc overexpression significantly decreased manganese superoxide dismutase expression and increased plasminogen activator inhibitor-1 expression in high-glucose conditions, which were recovered by SIRT1 overexpression. Moreover, compared to streptozotocin-induced wild-type diabetic mice, endothelium-specific SIRT1 transgenic diabetic mice had decreased p66Shc expression at both the mRNA and the protein levels, improved endothelial function, and reduced accumulation of nitrotyrosine and 8-OHdG (markers of oxidative stress). We further found that SIRT1 was able to bind to the p66Shc promoter (-508 bp to -250 bp), resulting in a decrease in the acetylation of histone H3 bound to the p66Shc promoter region. Our findings indicate that repression of p66Shc expression by SIRT1 contributes to the protection of hyperglycemia-induced endothelial dysfunction.

• Netrin-1 Prevents Ischemia/Reperfusion-induced Myocardial Infarction via a DCC/ERK1/2/eNOS(s1177)/NO/DCC Feed-forward Mechanism.

  Authors: Jun Zhang, Hua Cai

  Journal of molecular and cellular cardiology. 12/2009;

  We have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO(*)) production via a DCC-ERK1/2 dependent mechanism. In view of the well-establishedWe have recently shown that a novel endothelial mitogen netrin-1 potently stimulates nitric oxide (NO(*)) production via a DCC-ERK1/2 dependent mechanism. In view of the well-established cardioprotective role of NO(*), the present study investigated whether netrin-1 is cardioprotective via NO(*) signaling in the heart. Netrin-1 receptor DCC was abundantly expressed in the C57BL/6J mouse hearts. Perfusion of heart with netrin-1 (100 ng/mL) using a Langendorff system significantly increased NO(*) production. Under ischemia/reperfusion (I/R), netrin-1 induced a substantial reduction in infarct size (21.8+/-4.9% from 42.5+/-3.6% in the controls), which was accompanied by an augmented production of NO(*). Pre-perfusion with DCC-antibody, U0126 (MEK1/2 inhibitor), L-NAME or PTIO (NO(*) scavenger) attenuated protective effects of netrin-1 on infarct size and NO(*) production, indicating upstream roles of DCC and ERK1/2 in NO(*) production, as well as an essential role of NO(*) in cardioprotection. Netrin-1 induced reduction in infarct size was significantly attenuated in DCC+/-mice, confirming an intermediate role of DCC. In additional experiments we found netrin-1 increased ERK1/2 and eNOS(s1177) phosphorylation, and DCC protein expression, which was diminished by I/R. Furthermore, netrin-1-induced DCC upregulation was NO(*) and ERK1/2-dependent, implicating a feed-forward mechanism. DAF-AM staining revealed enhanced NO(*) production in both cardiac endothelial cells (ECs) and myocytes. In primarily isolated cardiomyocytes, netrin-1 also increased NO(*) production, DCC abundance and ERK1/2 phosphorylation. Of note, cardiac apoptosis was significantly attenuated by netrin-1, which was reversed by DCC-antibody, U0126, L-NAME or PTIO. In summary, our data clearly demonstrate that netrin-1 potently protects the heart from I/R injury by stimulating NO(*) production from cardiac ECs and myocytes. This potent effect is mediated by a DCC/ERK1/2/eNOS(s1177)/NO(*)/DCC feed-forward mechanism in both cell types.

• Targeted detoxification of selected reactive oxygen species in the vascular endothelium.

  Authors: Vladimir V Shuvaev, Melpo Christofidou-Solomidou, Faiz Bhora, Karine Laude, Hua Cai, Sergey Dikalov, Evguenia Arguiri, Charalambos C Solomides, Steven M Albelda, David G Harrison, Vladimir R Muzykantov

  The Journal of pharmacology and experimental therapeutics. 09/2009;

  Oxidative stress underlies diverse vascular diseases, but its management remains elusive, in part due to our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites andOxidative stress underlies diverse vascular diseases, but its management remains elusive, in part due to our inability to selectively detoxify reactive oxygen species (ROS) in pathological sites and limited understanding which species need to be eliminated. The antioxidant enzymes (AOE) superoxide dismutase (SOD) and catalase (which decompose O2(.-) and H2O2, respectively) conjugated with an antibody to Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) bind to endothelial cells and alleviate oxidative stress in cell culture models. Here, we studied the effects of these antioxidant conjugates in mouse models of vascular oxidative stress. Anti-PECAM/catalase and anti-PECAM/SOD conjugates, in contrast to control IgG/AOE conjugates, accumulated in the lungs and vascularized organs after intravenous injection in wild type, but not PECAM KO mice. Anti-PECAM/catalase, but not anti-PECAM/SOD, protected mice from lung injury induced by H2O2 produced by glucose oxidase deposited in the pulmonary vasculature. Anti-PECAM/catalase also reduced alveolar edema and attenuated decline in arterial oxygen in mice that underwent unilateral lung ischemia/reperfusion, while anti-PECAM/SOD was not effective, implying key role of H2O2 in tissue damage in this pathology. In contrast, anti-PECAM/SOD, but not anti-PECAM/catalase prevented oxidation of tetrahydrobiopterin (BH4) and normalized vasoreactivity in the vessels of mice rendered hypertensive by pre-treatment with angiotensin-II. This outcome agrees with reports implicating superoxide and peroxynitrite in altered endothelium-dependent vasodilatation in hypertension. Therefore, the use of endothelial cell-targeted antioxidants identifies the key specific species of ROS involved in various forms of vascular disease and holds promise for the mechanistically-tailored treatment of these pathologies.

• Endothelium-specific overexpression of human IC53 downregulates eNOS activity and elevates systolic blood pressure in mice.

  Authors: Ming-Lei Zhuo, Yue Huang, Jing-Zhou Chen, Li-Hong Sun, Rui-Feng Yang, Hou-Zao Chen, Xiang Lv, Hong-Liang Li, Yu-Sheng Wei, Guang Liu, Ran Zhang, Tie-Min Ma, Hua Cai, Ru-Tai Hui, De-Pei Liu, Chih-Chuan Liang

  Cardiovascular research. 07/2009;

  AIM: Hypertension is one of the major risk factors for cardiovascular diseases. Endothelial cells (ECs) exert important functions in the regulation of blood pressure. A novel gene, IC53, as anAIM: Hypertension is one of the major risk factors for cardiovascular diseases. Endothelial cells (ECs) exert important functions in the regulation of blood pressure. A novel gene, IC53, as an isoform of the cyclin-dependent kinase (CDK) binding protein gene C53, is mainly expressed in vascular endothelial cells and is upregulated in the failing heart of rats. Overexpression of IC53 promotes proliferation of endothelial cells. To examine whether IC53 plays a role in the regulation of vascular tone and blood pressure, we constructed a transgenic (tg) mouse model of the IC53 gene and studied its phenotypes relevant to vascular function. METHODS AND RESULTS: IC53 cDNA was cloned from a human aorta cDNA library. Using the endothelium-specific VE-cadherin promoter, we constructed transgenic mice in which IC53 was specifically overexpressed in vascular endothelia and found that the transgenic mice exhibit elevated systolic blood pressure in contrast to the wild-type (wt) controls. Further studies revealed impaired endothelium-dependent vasodilation, reduced nitric oxide production and decreased endothelial nitric oxide synthase (eNOS) expression and activity in the tg mice. Inhibition of IC53 in human umbilical vein endothelial cells induces upregulation of eNOS activity. CONCLUSIONS: Our results indicate that IC53 participates in the regulation of vascular homeostasis. Endothelium-specific overexpression of IC53 is associated with elevated systolic blood pressure, which may be in part attributed to downregulation of eNOS signaling.

• Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells.

  Authors: Gang Lu, Haipeng Sun, Pengxiang She, Ji-Youn Youn, Sarah Warburton, Peipei Ping, Thomas M Vondriska, Hua Cai, Christopher J Lynch, Yibin Wang

  The Journal of clinical investigation. 06/2009;

  The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acidsThe branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

• Aminoguanidine inhibits aortic hydrogen peroxide production, VSMC NOX activity and hypercontractility in diabetic mice.

  Authors: Jeong-Ho Oak, Ji-Youn Youn, Hua Cai

  Cardiovascular diabetology. 01/2009; 8:65.

  Dysfunctionally uncoupled endothelial nitric oxide synthase (eNOS) is involved in producing reactive oxygen species (ROS) in the diabetic endothelium. The present study investigated whetherDysfunctionally uncoupled endothelial nitric oxide synthase (eNOS) is involved in producing reactive oxygen species (ROS) in the diabetic endothelium. The present study investigated whether anti-diabetes drug Aminoguanidine (AG) has any effect on eNOS function and vascular oxidant stress. Blood glucose levels were increased to 452.0 +/- 15.1 mg/dl in STZ-treated male C57BL/6J mice (148.4 +/- 3.2 mg/dl in untreated controls). Aortic productions of NO* and O(2)*- were measured specifically and sensitively using electron spin resonance. Diabetic mice had a marked increase in aortic O(2)*- production. Aortic hydrogen peroxide (H(2)O(2)) production was also increased in diabetic aortas and significantly attenuated by AG. AG however had only a marginal effect in reducing aortic O(2)*- production, which corresponded to a minimal effect in improving aortic nitric oxide (NO*) bioavailability. The endothelium-dependent vasodilatation however was modestly but significantly improved by AG, likely consequent to AG-induced reduction in hyper-contractility. NAD(P)H oxidase (NOX)-dependent O(2)*- production was completely attenuated by AG in endothelium-denuded diabetic aortas. In summary, despite that AG is not an effective eNOS recoupling agent presumably consequent to its ineffectiveness in preventing endothelial NOX activation, it is inhibitory of aortic H(2)O(2) production, VSMC NOX activity, and hypercontractility in diabetes.

• An Ezrin/Calpain/PI3K/AMPK/eNOSs1179 Signaling Cascade Mediating VEGF-Dependent Endothelial Nitric Oxide Production.

  Authors: Ji-Youn Youn, Ting Wang, Hua Cai

  Circulation research. 12/2008;

  Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) inducedCalpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO., specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)s1179 phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO.. These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOSs1179 was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology.

• Endothelium-specific Overexpression of Class III Deacetylase SIRT1 Decreases Atherosclerosis in Apolipoprotein E-Deficient Mice.

  Authors: Qing-Jun Zhang, Zhao Wang, Hou-Zao Chen, Shuang Zhou, Wei Zheng, Guang Liu, Yu-Sheng Wei, Hua Cai, De-Pei Liu, Chih-Chuan Liang

  Cardiovascular research. 09/2008;

  AIMS: Hazardous environmental and genetic factors can damage endothelial cells to induce atherosclerotic vascular disease. Recent studies suggest that class III deacetylase SIRT1 may promote cellAIMS: Hazardous environmental and genetic factors can damage endothelial cells to induce atherosclerotic vascular disease. Recent studies suggest that class III deacetylase SIRT1 may promote cell survival via novel antioxidative mechanisms. The current study tested the hypothesis that SIRT1, specifically overexpressed in the endothelium, is atheroprotective. METHODS: Human umbilical vein endothelial cells (HUVECs) were used to study effects of oxidized LDL on SIRT1 expression. Endothelial cell-specific SIRT1 transgenic (SIRT1-Tg) mice were used to study the effect of SIRT1 on aortic vascular tone. SIRT1-Tg mice were crossed with apolipoprotein E null (apoE(-/-)) mice to obtain SIRT1-Tg/apoE(-/-) mice for analysis of atherogenesis in the presence of endothelial overexpression of SIRT1. RESULTS: SIRT1 expression in HUVECs was increased by treatment with oxidative LDL. Adenoviral-mediated overexpression of SIRT1 was protective of apoptosis of HUVECs. Calorie restriction increased, whereas high-fat diet decreased, SIRT1 expression in mouse aortas. In endothelial cell-specific SIRT1 transgenic (SIRT1-Tg) mice, high fat-induced impairment in endothelium-dependent vasorelaxation was improved compared to that of wild-type littermates. This was accompanied by an upregualtion of aortic endothelial nitric oxide synthase expression in the SIRT1-Tg mice. The SIRT1-Tg/apoE(-/-) mice had less atherosclerotic lesions compared to apoE(-/-) controls, without affecting blood lipids and glucose levels. CONCLUSIONS: These results suggest that endothelium-specific SIRT1 overexpression likely suppresses atherogenesis via improving endothelial cell survival and function.

• CaM Kinase II-dependent pathophysiological signalling in endothelial cells.

  Authors: Hua Cai, Depei Liu, Joe G N Garcia

  Cardiovascular research. 02/2008; 77(1):30-4.

  Calcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cellCalcium/calmodulin-dependent protein kinase II (CaM Kinase II) is a known modulator of cardiac pathophysiology. The present review uniquely focuses on novel CaM Kinase II-mediated endothelial cell signalling which, under pathophysiological conditions, may indirectly modulate cardiac functions via alterations in endothelial or endocardial responses. CaM Kinase II has four different isoforms and various splicing variants for each isoform. The endothelial cell CaM Kinase II isoforms are sensitive to KN93 and a threonine 286-mutated inhibitory peptide. In macrovascular endothelial cells derived from aortas, CaM Kinase II mediates redox-sensitive upregulation of endothelial nitric oxide synthase (eNOS) gene expression by hydrogen peroxide (H2O2) and oscillatory shear stress, and a rapid activation of eNOS in response to bradykinin. In endothelial cells derived from lung microvessels, CaM Kinase II mediates barrier dysfunction, particularly when activated by thrombin. In brain capillary endothelial cells, CaM Kinase II lies upstream of voltage-gated potassium channels and hypoxia-induced cell swelling. In both macrovascular and microvascular endothelial cells, CaM Kinase II mediates actin cytoskeleton reorganization via distinct p38 MAPK/HSP27 and ERK1/2/MLCK signalling pathways, respectively. Although understanding of endothelium-specific CaM Kinase II signalling is nascent, data accumulated so far have demonstrated a potentially significant role of CaM Kinase II in endothelial cell pathophysiology.

• Mitochondrial pathophysiology, reactive oxygen species, and cardiovascular diseases.

  Authors: Ling Gao, Karine Laude, Hua Cai

  The Veterinary clinics of North America. Small animal practice. 02/2008; 38(1):137-55, vi.

  This article discusses mitochondrial pathophysiology, reactive oxygen species, and cardiovascular diseases. Mitochondrial respiratory chains are responsible for energy metabolism/ATP productionThis article discusses mitochondrial pathophysiology, reactive oxygen species, and cardiovascular diseases. Mitochondrial respiratory chains are responsible for energy metabolism/ATP production through the tricyclic antidepressant cycle, coupling of oxidative phosphorylation, and electron transfer. The mitochondrion produces reactive oxygen species as "side products" of respiration. The mitochondrial derived reactive oxygen species is involved in the pathogenesis of various clinical disorders including heart failure, hypoxia, ischemia/reperfusion injury, diabetes, neurodegenerative diseases, and the physiologic process of aging. Observational and mechanistical studies of these pathologic roles of mitochondria are discussed in depth in this article.

• Targeted cardiac overexpression of A20 improves left ventricular performance and reduces compensatory hypertrophy after myocardial infarction.

  Authors: Hong-Liang Li, Ming-Lei Zhuo, Dong Wang, Ai-Bing Wang, Hua Cai, Li-Hong Sun, Qinglin Yang, Yue Huang, Yu-Sheng Wei, Peter P Liu, De-Pei Liu, Chih-Chuan Liang

  Circulation. 05/2007; 115(14):1885-94.

  BACKGROUND: A20 was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells. As an inhibitor of nuclear factor-kappaB signaling, A20 protectsBACKGROUND: A20 was originally characterized as a tumor necrosis factor-inducible gene in human umbilical vein endothelial cells. As an inhibitor of nuclear factor-kappaB signaling, A20 protects against apoptosis, inflammation, and cardiac hypertrophy. In the present study, we tested the hypothesis that cardiac-specific overexpression of A20 could protect the heart from myocardial infarction. METHODS AND RESULTS: We investigated the role of constitutive human A20 expression in acute myocardial infarction using a transgenic model. Transgenic mice containing the human A20 gene under the control of the alpha-myosin heavy chain promoter were constructed. Myocardial infarction was produced by coronary ligation in A20 transgenic mice and control animals. The extent of infarction was then quantified by 2-dimensional and M-mode echocardiography and by molecular and pathological analyses of heart samples in infarct and remote heart regions 7 days after myocardial infarction. Constitutive overexpression of A20 in the murine heart resulted in attenuated infarct size and improved cardiac function 7 days after myocardial infarction. Significantly, we found a decrease in nuclear factor-kappaB signaling and apoptosis, as well as proinflammatory response, cardiac remodeling, and interstitial fibrosis, in noninfarct regions in the hearts of constitutive A20-expressing animals compared with control animals. CONCLUSIONS: Cardiac-specific overexpression of A20 improves cardiac function and inhibits cardiac remodeling, apoptosis, inflammation, and fibrosis after acute myocardial infarction.

• Attenuation of angiotensin II signaling recouples eNOS and inhibits nonendothelial NOX activity in diabetic mice.

  Authors: Jeong-Ho Oak, Hua Cai

  Diabetes. 02/2007; 56(1):118-26.

  Angiotensin II (Ang II) levels are increased in patients with diabetes, but mechanisms underlying its contribution to diabetic vascular diseases are incompletely understood. We recently reported thatAngiotensin II (Ang II) levels are increased in patients with diabetes, but mechanisms underlying its contribution to diabetic vascular diseases are incompletely understood. We recently reported that in aortic endothelial cells, Ang II induces endothelial nitric oxide synthase (eNOS) uncoupling to produce superoxide (O(2)*(-)) rather than nitric oxide (NO*), upon loss of the tetrahydrobiopterin (H(4)B) salvage enzyme dihydrofolate reductase (DHFR). Here, we found that streptozotocin-induced diabetic mice had a marked increase in aortic O(2)*(-) production, which was inhibited by N-nitro-l-arginine methyl ester hydrochloride, indicating uncoupling of eNOS. Ang II receptor type 1 blocker candesartan or ACE inhibitor captopril markedly attenuated eNOS-derived O(2)*(-) and hydrogen peroxide production while augmenting NO* bioavailability in diabetic aortas, implicating recoupling of eNOS. O(2)*(-) and NO* production were characteristically and quantitatively measured by electron spin resonance. DHFR expression was decreased in diabetic aortas but significantly restored by candesartan or captopril. Either also improved vascular H(4)B content and endothelium-dependent vasorelaxation in diabetes. Rac1-dependent NAD(P)H oxidase (NOX) activity was more than doubled in the endothelium-denuded diabetic aortas but was attenuated by candesartan or captopril, indicating that NOX remains active in nonendothelial vascular tissues, although uncoupled eNOS is responsible for endothelial production of O(2)*(-). These data demonstrate a novel role of Ang II in diabetic uncoupling of eNOS and that Ang II-targeted therapy improves endothelial function via the novel mechanism of recoupling eNOS. Dual effectiveness on uncoupled eNOS and NOX may explain the high efficacy of Ang II antagonists in restoring endothelial function.

• Detection of reactive oxygen species and nitric oxide in vascular cells and tissues: comparison of sensitivity and specificity.

  Authors: Hua Cai, Sergey Dikalov, Kathy K Griendling, David G Harrison

  Methods in molecular medicine. 02/2007; 139:293-312.

  Reactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabeticReactive oxygen and nitrogen species are thought to contribute to pathogenesis of many cardiovascular diseases including hypertension, atherosclerosis, restenosis, heart failure, and diabetic vascular complications. Some of these reactive oxygen species also play an important role in vascular signaling. In this chapter, we describe various techniques that we have successfully employed to reliably measure superoxide and hydrogen peroxide. Because reactive oxygen species are capable of rapidly inactivating nitric oxide and because endothelial function characterized by nitric oxide bioavailability is an important indicator of vascular health, we have also included novel techniques capable of directly measuring nitric oxide radical from vascular cells and tissues.

• Netrin-1 induces angiogenesis via a DCC-dependent ERK1/2-eNOS feed-forward mechanism.

  Authors: Andrew Nguyen, Hua Cai

  Proceedings of the National Academy of Sciences of the United States of America. 05/2006; 103(17):6530-5.

  Netrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase inNetrin-1 is critical for axonal pathfinding which shares similarities with formation of vascular network. Here we report that netrin-1 induction of angiogenesis is mediated by an increase in endothelial nitric oxide (NO*) production, which occurs via a DCC-dependent, ERK1/2-eNOS feed-forward mechanism. Exposure of mature aortic endothelial cells to netrin-1 resulted in a potent, dose-dependent increase in NO* production, detected by electron spin resonance. Scavenging NO* with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) abolished netrin-1 stimulated angiogenesis. Netrin-1-stimulated NO* production or angiogenesis was inhibited by DCC antibody, DCC small interfering RNA (siRNA), specific inhibitors (PD98059, U0126), or siRNAs for MEK1/2. PTIO attenuated ERK1/2 phosphorylation, indicating a feed-forward mechanism. Netrin-1 induced a time-dependent phosphorylation of eNOS(s1179, s116) and a rapid dephosphorylation of eNOS(t497). Only eNOS(s1179) was sensitive to U0126 or PTIO. These data characterized a mechanism whereby netrin-1 promotes angiogenesis, which may broadly relate to cardiovascular, neuronal and cancer physiology.

• Ion-trap tandem mass spectrometric analysis of Amadori-glycated phosphatidylethanolamine in human plasma with or without diabetes.

  Authors: Kiyotaka Nakagawa, Jeong-Ho Oak, Ohki Higuchi, Tsuyoshi Tsuzuki, Shinichi Oikawa, Haruhisa Otani, Masatoshi Mune, Hua Cai, Teruo Miyazawa

  Journal of lipid research. 12/2005; 46(11):2514-24.

  Peroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabeticPeroxidized phospholipid-mediated cytotoxicity is involved in the pathophysiology of diseases [i.e., an abnormal increase of phosphatidylcholine hydroperoxide (PCOOH) in plasma of type 2 diabetic patients]. The PCOOH accumulation may relate to Amadori-glycated phosphatidylethanolamine (Amadori-PE; deoxy-D-fructosyl phosphatidylethanolamine), because Amadori-PE causes oxidative stress. However, the occurrence of lipid glycation products, including Amadori-PE, in vivo is still unclear. Consequently, we developed an analysis method of Amadori-PE using a quadrupole/linear ion-trap mass spectrometer, the Applied Biosystems QTRAP. In positive ion mode, collision-induced dissociation of Amadori-PE produced a well-characterized diglyceride ion ([M+H-303]+) permitting neutral loss scanning and multiple reaction monitoring (MRM). When lipid extract from diabetic plasma was infused directly into the QTRAP, Amadori-PE molecular species could be screened out by neutral loss scanning. Interfacing liquid chromatography with QTRAP mass spectrometry enabled the separation and determination of predominant plasma Amadori-PE species with sensitivity of approximately 0.1 pmol/injection in MRM. The plasma Amadori-PE level was 0.08 mol% of total PE in healthy subjects and 0.15-0.29 mol% in diabetic patients. Furthermore, plasma Amadori-PE levels were positively correlated with PCOOH (a maker for oxidative stress). These results show the involvement between lipid glycation and lipid peroxidation in diabetes pathogenesis.

• Hydrogen peroxide regulation of endothelial function: origins, mechanisms, and consequences.

  Authors: Hua Cai

  Cardiovascular research. 11/2005; 68(1):26-36.

  Increased production of reactive oxygen species (ROS) has been implicated in the pathogenesis of cardiovascular diseases. Enzymatic systems such as the mitochondrial respiratory chain, vascularIncreased production of reactive oxygen species (ROS) has been implicated in the pathogenesis of cardiovascular diseases. Enzymatic systems such as the mitochondrial respiratory chain, vascular NAD(P)H oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS) produce superoxide anion (O2*-) in vascular cells. While some O2(*-) rapidly degrades by reacting with nitric oxide (NO*), the O2*- signal preserved by dismutation into hydrogen peroxide (H2O2) exerts prolonged signaling effects. This review focuses on patterns and mechanisms whereby H2O2 modulates different aspects of endothelial cell function including endothelial cell growth and proliferation, endothelial apoptosis, endothelium-dependent vasorelaxation, endothelial cytoskeletal reorganization and barrier dysfunction, endothelial inflammatory responses, and endothelium-regulated vascular remodeling. These modulations of endothelial cell function may at least partially underlie H2O2 contribution to the development of vascular disease.

• Regulation of xanthine oxidoreductase protein expression by hydrogen peroxide and calcium.

  Authors: J Scott McNally, Archana Saxena, Hua Cai, Sergey Dikalov, David G Harrison

  Arteriosclerosis, thrombosis, and vascular biology. 09/2005; 25(8):1623-8.

  OBJECTIVE: We have previously demonstrated that endothelial xanthine oxidase (XO) levels are dependent on the NADPH oxidase. We postulated that H2O2 may modulate the irreversible conversion ofOBJECTIVE: We have previously demonstrated that endothelial xanthine oxidase (XO) levels are dependent on the NADPH oxidase. We postulated that H2O2 may modulate the irreversible conversion of xanthine dehydrogenase (XDH) to XO and sought to examine mechanisms involved. METHODS AND RESULTS: H2O2 (100 micromol/L) decreased bovine aortic endothelial cell (BAEC) XDH protein expression, and metabolic labeling studies indicated that H2O2 stimulated conversion of XDH to XO. The decline in XDH was mimicked by the reactive oxygen species (ROS) generating compounds SIN-1 and Menadione, as well as by stimulating BAECs with angiotensin II (200 nmol/L). BAPTA-AM prevented the decline in XDH by H2O2, indicating that it was calcium-dependent. In keeping with calcium acting downstream of H2O2, the calcium ionophore A23187 (1 micromol/L) caused XDH-to-XO conversion, and this was not prevented by the antioxidants. In addition, XDH-to-XO conversion was blocked by 2-APB and NO donors and induced by thapsigargin and M-3M3FBS, implicating phospholipase C and endoplasmic reticulum calcium stores in this process. CONCLUSIONS: Endothelial XO and XDH expression are strongly dependent on H2O2 and calcium. Stimulation of XDH conversion to XO may represent a feed-forward mechanism whereby H2O2 can stimulate further production of ROS.

• Endothelial dihydrofolate reductase: critical for nitric oxide bioavailability and role in angiotensin II uncoupling of endothelial nitric oxide synthase.

  Authors: Karel Chalupsky, Hua Cai

  Proceedings of the National Academy of Sciences of the United States of America. 07/2005; 102(25):9056-61.

  Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O2*-). H4B was foundRecent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H4B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (O2*-). H4B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H4B and nitric oxide (NO.) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H4B deficiency that was mediated by H2O2-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial O2*- production, which was abolished by eNOS inhibitor N-nitro-L-arginine-methyl ester or H2O2 scavenger polyethylene glycol-conjugated catalase, strongly suggesting H2O2-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of O2* (Rac1 inhibitor NSC 23766 but not an N-nitro-L-arginine-methyl ester-attenuated ESR O2*- signal at 30 min) in response to angiotensin II, preceding a second peak in O2*- production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO. production and diminished eNOS production of O2*- in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H4B and NO. bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H2O2 down-regulates DHFR expression in response to angiotensin II, resulting in H4B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.

• NAD(P)H oxidase-dependent self-propagation of hydrogen peroxide and vascular disease.

  Authors: Hua Cai

  Circulation research. 05/2005; 96(8):818-22.

  Excessive production of reactive oxygen species in the vasculature contributes to cardiovascular pathogenesis. Among biologically relevant and abundant reactive oxygen species, superoxide (O2*-) andExcessive production of reactive oxygen species in the vasculature contributes to cardiovascular pathogenesis. Among biologically relevant and abundant reactive oxygen species, superoxide (O2*-) and hydrogen peroxide (H2O2) appear most important in redox signaling. Whereas O2*- predominantly induces endothelial dysfunction by rapidly inactivating nitric oxide (NO*), H2O2 influences different aspects of endothelial cell function via complex mechanisms. This review discusses recent advances establishing a critical role of H2O2 in the development of vascular disease, in particular, atherosclerosis, and mechanisms whereby vascular NAD(P)H oxidase-derived H2O2 amplifies its own production. Recent studies have shown that H2O2 stimulates reactive oxygen species production via enhanced intracellular iron uptake, mitochondrial damage, and sources of vascular NAD(P)H oxidases, xanthine oxidase, and uncoupled endothelial nitric oxide synthase (eNOS). This self-propagating phenomenon likely prolongs H2O2-dependent pathological signaling in vascular cells, thus contributing to vascular disease development. The latest progress on Nox functions in vascular cells is also discussed [Nox for NAD(P)H oxidases, representing a family of novel NAD(P)H oxidases].