Alex F. Chen

Endothelin-1 increases vascular superoxide via endothelin (A)-NADPH oxidase pathway in low-renin hypertension

Background—Angiotensin II–induced hypertension is associated with NAD(P)H oxidase–dependent superoxide production in the vessel wall. Vascular superoxide level is also increased in deoxycorticosterone acetate (DOCA)–salt hypertension, which is associated with a markedly depressed plasma renin activity because of sodium retention. However, the mechanisms underlying superoxide production in low-renin hypertension are undefined. Methods and Results—This study investigated (1) whether and how endothelin-1 (ET-1), which is increased in DOCA-salt hypertensive rats, contributes to arterial superoxide generation and (2) the effect of gene transfer of manganese superoxide dismutase and endothelial nitric oxide synthase. Both superoxide and ET-1 levels were significantly elevated in carotid arteries of DOCA-salt rats compared with that of the sham-operated controls. ET-1 concentration-dependently stimulated superoxide production in vitro in carotid arteries of normotensive rats. The increase in arterial superoxide in both ET-1–treated normotensive and DOCA-salt rats was reversed by a selective ETA receptor antagonist, ABT-627, the flavoprotein inhibitor diphenyleneiodonium, and the NADPH oxidase inhibitor apocynin but not by the nitric oxide synthase inhibitor N&ohgr;-l-arginine methyl ester or the xanthine oxidase inhibitor allopurinol. Furthermore, in vivo blockade of ETA receptors significantly reduced arterial superoxide levels, with a concomitant decrease of systolic blood pressure in DOCA-salt rats. Ex vivo gene transfer of manganese superoxide dismutase or endothelial nitric oxide synthase also suppressed superoxide levels in carotid arteries of DOCA-salt rats. Conclusions—These findings suggest that ET-1 augments vascular superoxide production at least in part via an ETA/NADPH oxidase pathway in low-renin mineralocorticoid hypertension.

MicroRNA-34a induces endothelial progenitor cell senescence and impedes its angiogenesis via suppressing silent information regulator 1

Endothelial progenitor cells (EPCs) play an important role in angiogenesis, which is essential for numerous physiological processes as well as tumor growth. Several microRNAs (miRNAs) have been reported to be involved in angiogenesis. MiR-34a, recently reported as a tumor suppressor, has been found to target silent information regulator 1 (Sirt1), leading to cell cycle arrest or apoptosis. However, the role of miR-34a in EPC-mediated angiogenesis was unknown. The present study tested the hypothesis that miR-34a inhibits EPC-mediated angiogenesis by inducing senescence via suppressing Sirt1. Bone marrow-derived EPCs from adult male Sprague-Dawley rats were used. Results of flow cytometry showed that EPCs after 7 days of culture expressed both stem cell markers CD34 and CD133 and endothelial cell markers VEGFR-2 (flk-1) and VE-cadherin. MiR-34a was expressed in normal EPCs, and overexpression of miR-34a via its mimic transfection significantly increased its expression and impaired in vitro EPC angiogenesis. MiR-34a overexpression led to a significantly increased EPC senescence, paralleled with an approximately 40% Sirt1 reduction. Furthermore, knockdown of Sirt1 by its siRNA resulted in diminished EPC angiogenesis and increased senescence. Finally, overexpression of miR-34a increased the level of Sirt1 effector-acetylated forkhead box O transcription factors 1 (FoxO1), an effect mimicked in EPCs following Sirt1 knockdown. In conclusion, miR-34a impairs EPC-mediated angiogenesis by induction of senescence via inhibiting Sirt1.

Signature microRNA Expression Profile of Essential Hypertension and Its Novel Link to Human Cytomegalovirus Infection

Background— Essential hypertension has been recognized as a disease resulting from a combination of environmental and genetic factors. Recent studies demonstrated that microRNAs (miRNAs) are involved in cardiac hypertrophy and heart failure. However, little is known about the roles of miRNAs in essential hypertension. Methods and Results— Using microarray-based miRNA expression profiling, we compared the miRNA expressions in plasma samples from 13 hypertensive patients and 5 healthy control subjects. Twenty-seven miRNAs were found to be differentially expressed. The expressions of selected miRNAs (miR-296–5p, let-7e, and a human cytomegalovirus [HCMV]–encoded miRNA, hcmv-miR-UL112) were validated independently in plasma samples from 24 hypertensive patients and 22 control subjects. The absolute expression levels of hcmv-miR-UL112, miR-296–5p, and let-7e were further determined in 127 patients and 67 control subjects (fold changes are 2.5, 0.5, and 1.7 respectively; all P<0.0001). Additionally, we demonstrated that interferon regulatory factor 1 is a direct target of hcmv-miR-UL112. Increased HCMV seropositivity and quantitative titers were found in the hypertension group compared with the control group (52.7% versus 30.9%, P=0.0005; 1870 versus 54 copies per 1 mL plasma, P<0.0001). Seropositivity, log-transformed copies of HCMV, and hcmv-miR-UL112 were independently associated with an increased risk of hypertension (odds ratio, 2.48; 95% confidence interval, 1.48 to 4.15; P=0.0005; odds ratio, 1.97; 95% confidence interval, 1.58 to 2.46; P<0.0001; and odds ratio, 2.55; 95% confidence interval, 1.98 to 3.27; P<0.0001, respectively). Conclusions— We report for the first time a circulating miRNA profile for hypertensive patients and demonstrate a novel link between HCMV infection and essential hypertension. These findings may reveal important insights into the pathogenesis of essential hypertension. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00420784.

Nitric oxide: a newly discovered function on wound healing

AbstractWound healing impairment represents a particularly challenging clinical problem to which no efficacious treatment regimens currently exist. The factors ensuring appropriate intercellular communication during wound repair are not completely understood. Although protein-type mediators are well-established players in this process, emerging evidence from both animal and human studies indicates that nitric oxide (NO) plays a key role in wound repair. The beneficial effects of NO on wound repair may be attributed to its functional influences on angiogenesis, inflammation, cell proliferation, matrix deposition, and remodeling. Recent findings from in vitro and in vivo studies of NO on wound repair are summarized in this review. The unveiled novel mechanisms support the use of NO-containing agents and/or NO synthase gene therapy as new therapeutic regimens for impaired wound healing.

Leptin Induces Hypertrophy via Endothelin-1–Reactive Oxygen Species Pathway in Cultured Neonatal Rat Cardiomyocytes

Background—Obesity is a major risk factor for the development of cardiovascular disease. Emerging evidence indicates that leptin, a protein encoded by the obesity gene, is linked with cardiac hypertrophy in obese humans and directly induces cardiomyocyte hypertrophy in vitro. However, the mechanisms by which leptin induces cardiomyocyte hypertrophy are poorly understood. Methods and Results—This study investigated how leptin contributes to cardiomyocyte hypertrophy. Cultured neonatal rat cardiomyocytes were used to evaluate the effects of leptin on hypertrophy. Both endothelin-1 (ET-1) and reactive oxygen species (ROS) levels were elevated in a concentration-dependent manner in cardiomyocytes treated with leptin for 4 hours compared with those cells without leptin treatment. ET-1 stimulated ROS production in a concentration-dependent manner in cardiomyocytes. The augmentation of ROS levels in cardiomyocytes treated with both leptin and ET-1 was reversed by a selective ETA receptor antagonist, ABT-627, and catalase, a hydrogen peroxide–decomposing enzyme. After treatment for 72 hours, leptin or ET-1 concentration-dependently increased total RNA levels, cell surface areas, and protein synthesis in cardiomyocytes, all of which were significantly inhibited by ABT-627 or catalase treatment. ConclusionsThese findings indicate that leptin elevates ET-1 and ROS levels, resulting in hypertrophy of cultured neonatal rat cardiac myocytes. The ET-1–ETA–ROS pathway may be involved in cardiomyocyte hypertrophy induced by leptin. ETA receptor antagonists and antioxidant therapy may provide an effective means of ameliorating cardiac dysfunction in obese humans.

Curcumin-loaded poly(ε-caprolactone) nanofibres: Diabetic wound dressing with anti-oxidant and anti-inflammatory properties

1 Curcumin is a naturally occurring poly‐phenolic compound with a broad range of favourable biological functions, including anti‐cancer, anti‐oxidant and anti‐inflammatory activities. The low bioavailability and in vivo stability of curcumin require the development of suitable carrier vehicles to deliver the molecule in a sustained manner at therapeutic levels. 2 In the present study, we investigated the feasibility and potential of poly(caprolactone) (PCL) nanofibres as a delivery vehicle for curcumin for wound healing applications. By optimizing the electrospinning parameters, bead‐free curcumin‐loaded PCL nanofibres were developed. 3 The fibres showed sustained release of curcumin for 72 h and could be made to deliver a dose much lower than the reported cytotoxic concentration while remaining bioactive. Human foreskin fibroblast cells (HFF‐1) showed more than 70% viability on curcumin‐loaded nanofibres. 4 The anti‐oxidant activity of curcumin‐loaded nanofibres was demonstrated using an oxygen radical absorbance capacity (ORAC) assay and by the ability of the fibres to maintain the viability of HFF‐1 cells under conditions of oxidative stress. 5 The curcumin‐loaded nanofibres also reduced inflammatory induction, as evidenced by low levels of interleukin‐6 release from mouse monocyte–macrophages seeded onto the fibres following stimulation by Escherichia coli‐derived lipopolysaccharide. 6 The in vivo wound healing capability of the curcumin loaded PCL nanofibres was demonstrated by an increased rate of wound closure in a streptozotocin‐induced diabetic mice model. 7 These results demonstrate that the curcumin‐loaded PCL nanofibre matrix is bioactive and has potential as a wound dressing with anti‐oxidant and anti‐inflammatory properties.

Gene Transfer of Human Guanosine 5′-Triphosphate Cyclohydrolase I Restores Vascular Tetrahydrobiopterin Level and Endothelial Function in Low Renin Hypertension

Background—We recently reported that arterial superoxide (O2−) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH4), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O2−–induced vascular dysfunction. However, the interaction between O2− and BH4 on endothelial function and the underlying mechanisms are unknown. Methods and Results—The present study tested the hypothesis that BH4 deficiency due to ET-1–induced O2− leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O2− levels and decreased GTPCH I activity and BH4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ETA receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O2− and restored BH4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH4 levels, resulting in reduced O2− and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions—These results indicate that a BH4 deficiency resulting from ET-1–induced O2− via an ETA/NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH4 deficiency and endothelial dysfunction by reducing O2− in low renin mineralocorticoid hypertension.

Gene Therapy of Endothelial Nitric Oxide Synthase and Manganese Superoxide Dismutase Restores Delayed Wound Healing in Type 1 Diabetic Mice

Background—Nitric oxide (NO) deficiency contributes to diabetic wound healing impairment. The present study tested the hypothesis that increased cutaneous superoxide (O2−) levels in type 1 diabetic mice cause NO deficiency and delayed wound healing. Methods and Results—Wound healing was markedly delayed in streptozotocin-induced type 1 diabetic mice compared with the normal controls. There were significantly reduced levels of endothelial NO synthase (eNOS) protein and constitutive NOS activity in diabetic wounds, whereas O2− levels were markedly increased. A single regimen of cutaneous gene therapy of eNOS or manganese superoxide dismutase (MnSOD) restored such healing delay, with a concomitant suppression of wound O2− levels and augmentation of both eNOS protein and constitutive NOS activity. Gene therapy of MnSOD also increased cutaneous MnSOD activity. Cutaneous O2− levels were also increased in Ins2Akita diabetic mice. In vitro glucose treatment of cutaneous tissues from normal mice for 24 hours increased O2− levels in a concentration-dependent manner. The enhanced cutaneous O2− levels induced by high glucose in both normal and diabetic mice were abolished by the NADPH oxidase inhibitor apocynin and the protein kinase C inhibitor chelerythrine. Furthermore, ex vivo gene transfer of dominant-negative HA-tagged N17Rac1, which inhibits NADPH oxidase subunit Rac1, significantly inhibited cutaneous O2− formation induced by high glucose in both normal and Ins2Akita diabetic mice. Conclusions—These results indicate that hyperglycemia augments cutaneous O2− levels, at least in part, via NADPH oxidase and protein kinase C pathways, resulting in impaired wound healing in type 1 diabetic mice. Gene therapy strategies aimed at restoring cutaneous NO bioavailability may provide an effective means to ameliorate delayed diabetic wound healing.

Manganese superoxide dismutase expression in endothelial progenitor cells accelerates wound healing in diabetic mice

Amputation as a result of impaired wound healing is a serious complication of diabetes. Inadequate angiogenesis contributes to poor wound healing in diabetic patients. Endothelial progenitor cells (EPCs) normally augment angiogenesis and wound repair but are functionally impaired in diabetics. Here we report that decreased expression of manganese superoxide dismutase (MnSOD) in EPCs contributes to impaired would healing in a mouse model of type 2 diabetes. A decreased frequency of circulating EPCs was detected in type 2 diabetic (db/db) mice, and when isolated, these cells exhibited decreased expression and activity of MnSOD. Wound healing and angiogenesis were markedly delayed in diabetic mice compared with normal controls. For cell therapy, topical transplantation of EPCs onto excisional wounds in diabetic mice demonstrated that diabetic EPCs were less effective than normal EPCs at accelerating wound closure. Transplantation of diabetic EPCs after MnSOD gene therapy restored their ability to mediate angiogenesis and wound repair. Conversely, siRNA-mediated knockdown of MnSOD in normal EPCs reduced their activity in diabetic wound healing assays. Increasing the number of transplanted diabetic EPCs also improved the rate of wound closure. Our findings demonstrate that cell therapy using diabetic EPCs after ex vivo MnSOD gene transfer accelerates their ability to heal wounds in a mouse model of type 2 diabetes.

Airborne particulate matter selectively activates endoplasmic reticulum stress response in the lung and liver tissues

Recent studies have suggested a link between inhaled particulate matter (PM) exposure and increased mortality and morbidity associated with pulmonary and cardiovascular diseases. However, a precise understanding of the biological mechanism underlying PM-associated toxicity and pathogenesis remains elusive. Here, we investigated the impact of PM exposure in intracellular stress signaling pathways with animal models and cultured cells. Inhalation exposure of the mice to environmentally relevant fine particulate matter (aerodynamic diameter < 2.5 μm, PM(2.5)) induces endoplasmic reticulum (ER) stress and activation of unfolded protein response (UPR) in the lung and liver tissues as well as in the mouse macrophage cell line RAW264.7. Ambient PM(2.5) exposure activates double-strand RNA-activated protein kinase-like ER kinase (PERK), leading to phosphorylation of translation initiation factor eIF2α and induction of C/EBP homologous transcription factor CHOP/GADD153. Activation of PERK-mediated UPR pathway relies on the production of reactive oxygen species (ROS) and is critical for PM(2.5)-induced apoptosis. Furthermore, PM(2.5) exposure can activate ER stress sensor IRE1α, but it decreases the activity of IRE1α in splicing the mRNA encoding the UPR trans-activator X-box binding protein 1 (XBP1). Together, our study suggests that PM(2.5) exposure differentially activates the UPR branches, leading to ER stress-induced apoptosis through the PERK-eIF2α-CHOP UPR branch. This work provides novel insights into the cellular and molecular basis by which ambient PM(2.5) exposure elicits its cytotoxic effects that may be related to air pollution-associated pathogenesis.