Mechanisms of obesity-induced metabolic and vascular dysfunctions.

Obesity has reached epidemic proportions and its prevalence is climbing. Obesity is characterized by hypertrophied adipocytes with a dysregulated adipokine secretion profile, increased recruitment of inflammatory cells, and impaired metabolic homeostasis that eventually results in the development of systemic insulin resistance, a phenotype of type 2 diabetes. Nitric oxide synthase (NOS) is an enzyme that converts L-arginine to nitric oxide (NO), which functions to maintain vascular and adipocyte homeostasis. Arginase is a ureohydrolase enzyme that competes with NOS for L-arginine. Arginase activity/expression is upregulated in obesity, which results in diminished bioavailability of NO, impairing both adipocyte and vascular endothelial cell function. Given the emerging role of NO in the regulation of adipocyte physiology and metabolic capacity, this review explores the interplay between arginase and NO, and their effect on the development of metabolic disorders, cardiovascular diseases, and mitochondrial dysfunction in obesity. A comprehensive understanding of the mechanisms involved in the development of obesity-induced metabolic and vascular dysfunction is necessary for the identification of more effective and tailored therapeutic avenues for their prevention and treatment.

The TNF-derived TIP peptide activates the epithelial sodium channel and ameliorates experimental nephrotoxic serum nephritis.

In mice, the initial stage of nephrotoxic serum-induced nephritis (NTN) mimics antibody-mediated human glomerulonephritis. Local immune deposits generate tumor necrosis factor (TNF), which activates pro-inflammatory pathways in glomerular endothelial cells (GECs) and podocytes. Because TNF receptors mediate antibacterial defense, existing anti-TNF therapies can promote infection; however, we have previously demonstrated that different functional domains of TNF may have opposing effects. The TIP peptide mimics the lectin-like domain of TNF, and has been shown to blunt inflammation in acute lung injury without impairing TNF receptor-mediated antibacterial activity. We evaluated the impact of TIP peptide in NTN. Intraperitoneal administration of TIP peptide reduced inflammation, proteinuria, and blood urea nitrogen. The protective effect was blocked by the cyclooxygenase inhibitor indomethacin, indicating involvement of prostaglandins. Targeted glomerular delivery of TIP peptide improved pathology in moderate NTN and reduced mortality in severe NTN, indicating a local protective effect. We show that TIP peptide activates the epithelial sodium channel(ENaC), which is expressed by GEC, upon binding to the channel's α subunit. In vitro, TNF treatment of GEC activated pro-inflammatory pathways and decreased the generation of prostaglandin E2 and nitric oxide, which promote recovery from NTN. TIP peptide counteracted these effects. Despite the capacity of TIP peptide to activate ENaC, it did not increase mean arterial blood pressure in mice. In the later autologous phase of NTN, TIP peptide blunted the infiltration of Th17 cells. By countering the deleterious effects of TNF through direct actions in GEC, TIP peptide could provide a novel strategy to treat glomerular inflammation.

Hyperglycemia-impaired aortic vasorelaxation mediated through arginase elevation: Role of stress kinase pathways.

Diabetes-induced vascular endothelial dysfunction has been reported to involve hyperglycemia-induced increases in arginase activity. However, upstream mediators of this effect are not clear. Here, we have tested involvement of Rho kinase, ERK1/2 and p38 MAPK pathways in this process. Studies were performed with aortas isolated from wild type or hemizygous arginase 1 knockout (Arg1+/-) mice and bovine aortic endothelial cells exposed to high glucose (HG, 25 mmol/l) or normal glucose (NG, 5.5 mmol/l) conditions for different times. Effects of inhibitors of arginase, p38 MAPK, ERK1/2 or ROCK and ex vivo adenoviral delivery of active Arg1 and inactive (D128-Arg1) cDNA were also determined. Exposure in wild type aorta or endothelial cells to HG significantly increased arginase activity and Arg1 expression and impaired aortic relaxation. Transduction of wild type aorta with active Arg1 cDNA impaired vascular relaxation, whereas inactive Arg1 had no effect. The HG-induced vascular endothelial dysfunction was associated with increased phosphorylation (activation) of ERK1/2 and p38 MAPK. Pretreatment with inhibitors of ERK1/2, p38 MAPK, ROCK or arginase blocked HG-induced elevation of arginase activity and Arg1 expression and prevented the vascular dysfunction. Inhibition of ROCK blunted the HG-induced activation of ERK1/2 and p38 MAPK. In summary, activated ROCK and subsequent activation of ERK1/2 or p38 MAPK elevates arginase activity and Arg1 expression in hyperglycemic states. Targeting this pathway may provide an effective means for preventing diabetes/hyperglycemia-induced vascular endothelial dysfunction.

Arginase 1 promotes retinal neurovascular protection from ischemia through suppression of macrophage inflammatory responses.

The lack of effective therapies to limit neurovascular injury in ischemic retinopathy is a major clinical problem. This study aimed to examine the role of ureohydrolase enzyme, arginase 1 (A1), in retinal ischemia-reperfusion (IR) injury. A1 competes with nitric oxide synthase (NOS) for their common substrate L-arginine. A1-mediated L-arginine depletion reduces nitric oxide (NO) formation by NOS leading to vascular dysfunction when endothelial NOS is involved but prevents inflammatory injury when inducible NOS is involved. Studies were performed using wild-type (WT) mice, global A1+/- knockout (KO), endothelial-specific A1 KO, and myeloid-specific A1 KO mice subjected to retinal IR injury. Global as well as myeloid-specific A1 KO mice showed worsened IR-induced neuronal loss and retinal thinning. Deletion of A1 in endothelial cells had no effect, while treatment with PEGylated (PEG) A1 improved neuronal survival in WT mice. In addition, A1+/- KO mice showed worsened vascular injury manifested by increased acellular capillaries. Western blotting analysis of retinal tissue showed increased inflammatory and necroptotic markers with A1 deletion. In vitro experiments showed that macrophages lacking A1 exhibit increased inflammatory response upon LPS stimulation. PEG-A1 treatment dampened this inflammatory response and decreased the LPS-induced metabolic reprogramming. Moreover, intravitreal injection of A1 KO macrophages or systemic macrophage depletion with clodronate liposomes increased neuronal loss after IR injury. These results demonstrate that A1 reduces IR injury-induced retinal neurovascular degeneration via dampening macrophage inflammatory responses. Increasing A1 offers a novel strategy for limiting neurovascular injury and promoting macrophage-mediated repair.

Arginase: A Multifaceted Enzyme Important in Health and Disease.

The arginase enzyme developed in early life forms and was maintained during evolution. As the last step in the urea cycle, arginase cleaves l-arginine to form urea and l-ornithine. The urea cycle provides protection against excess ammonia, while l-ornithine is needed for cell proliferation, collagen formation, and other physiological functions. In mammals, increases in arginase activity have been linked to dysfunction and pathologies of the cardiovascular system, kidney, and central nervous system and also to dysfunction of the immune system and cancer. Two important aspects of the excessive activity of arginase may be involved in diseases. First, overly active arginase can reduce the supply of l-arginine needed for the production of nitric oxide (NO) by NO synthase. Second, too much l-ornithine can lead to structural problems in the vasculature, neuronal toxicity, and abnormal growth of tumor cells. Seminal studies have demonstrated that increased formation of reactive oxygen species and key inflammatory mediators promote this pathological elevation of arginase activity. Here, we review the involvement of arginase in diseases affecting the cardiovascular, renal, and central nervous system and cancer and discuss the value of therapies targeting the elevated activity of arginase.

Netrin-1 is a novel regulator of vascular endothelial function in diabetes.

BACKGROUND: Netrin-1, a secreted laminin-like protein identified as an axon guidance molecule, has been shown to be of critical importance in the cardiovascular system. Recent studies have revealed pro-angiogenic, anti-apoptotic and anti-inflammatory properties of netrin-1 as well as cardioprotective actions against myocardial injury in diabetic mice. AIM: To examine the role of netrin-1 in diabetes-and high glucose (HG)-induced vascular endothelial dysfunction (VED) using netrin-1 transgenic mice (Tg3) and cultured bovine aortic endothelial cells (BAEC). MAIN OUTCOME: Overexpression of netrin-1 prevented diabetes-induced VED in aorta from diabetic mice and netrin-1 treatment attenuated HG-induced impairment of nitric oxide synthase (NOS) function in BAECs. METHODS AND RESULTS: Experiments were performed in Tg3 and littermate control (WT) mice rendered diabetic with streptozotocin (STZ) and in BAECs treated with HG (25 mmol/L). Levels of netrin-1 and its receptor DCC, markers of inflammation and apoptosis and vascular function were assessed in aortas from diabetic and non-diabetic Tg3 and WT mice. Vascular netrin-1 in WT mice was reduced under diabetic conditions. Aortas from non-diabetic Tg3 and WT mice showed similar maximum endothelium-dependent relaxation (MEDR) (83% and 87%, respectively). MEDR was markedly impaired in aorta from diabetic WT mice (51%). This effect was significantly blunted in Tg3 diabetic aortas (70%). Improved vascular relaxation in Tg3 diabetic mice was associated with increased levels of phospho-ERK1/2 and reduced levels of oxidant stress, NFκB, COX-2, p16INK4A, cleaved caspase-3 and p16 and p53 mRNA. Netrin-1 treatment prevented the HG-induced decrease in NO production and elevation of oxidative stress and apoptosis in BAECs. CONCLUSIONS: Diabetes decreases aortic levels of netrin-1. However, overexpression of netrin-1 attenuates diabetes-induced VED and limits the reduction of NO levels, while increasing expression of p-ERK1/2, and suppressing oxidative stress and inflammatory and apoptotic processes. Enhancement of netrin-1 function may be a useful therapeutic means for preventing vascular dysfunction in diabetes.

Regulation of endothelial intracellular adenosine via adenosine kinase epigenetically modulates vascular inflammation.

The molecular mechanisms underlying vascular inflammation and associated inflammatory vascular diseases are not well defined. Here we show that endothelial intracellular adenosine and its key regulator adenosine kinase (ADK) play important roles in vascular inflammation. Pro-inflammatory stimuli lead to endothelial inflammation by increasing endothelial ADK expression, reducing the level of intracellular adenosine in endothelial cells, and activating the transmethylation pathway through increasing the association of ADK with S-adenosylhomocysteine (SAH) hydrolase (SAHH). Increasing intracellular adenosine by genetic ADK knockdown or exogenous adenosine reduces activation of the transmethylation pathway and attenuates the endothelial inflammatory response. In addition, loss of endothelial ADK in mice leads to reduced atherosclerosis and affords protection against ischemia/reperfusion injury of the cerebral cortex. Taken together, these results demonstrate that intracellular adenosine, which is controlled by the key molecular regulator ADK, influences endothelial inflammation and vascular inflammatory diseases.The molecular mechanisms underlying vascular inflammation are unclear. Here the authors show that pro-inflammatory stimuli lead to endothelial inflammation by increasing adenosine kinase expression, and that its knockdown in endothelial cells inhibits atherosclerosis and cerebral ischemic injury in mice.

Toll-like receptor 4 (TLR4) impairs nitric oxide contributing to Angiotensin II-induced cavernosal dysfunction.

AIM: Angiotensin II (AngII), a corpus cavernosum (CC) constrictor peptide, modulates Toll like receptor (TLR) expression, a key element of the innate immune system, contributing to impaired vascular function in pathological conditions. However, it is unknown whether TLR4 is involved in AngII-induced erectile dysfunction. In this study, we investigated whether TLR4 plays a role in cavernosal dysfunction caused by AngII upregulation. MATERIAL AND METHODS: Cavernosal smooth muscle cells (CSMC) from C57/BL6 mice were treated with AngII (0.1µM) or bacterial LPS (50ng/ml) for 12-24h and TLR4 expression was assessed. Mice were infused with AngII (90ng/min, 28days) and treated with anti-TLR4 antibody (0.1mg/daily, i.p.) for the last 14days of the treatment. CC tissue was used for functional studies and for Western blotting. Nitric Oxide Synthase (NOS) activity was measured by conversion of [3H]-l-arginine to [3H]-l-citrulline, systemic TNF-α levels by ELISA, and reactive oxygen species (ROS) by immunofluorescence. KEY FINDINGS: We report upregulation of TLR4 in CSMC following AngII or LPS stimulation. In AngII-infused mice, chronic treatment with anti-TLR4 antibody (28±2.1%) attenuates adrenergic CC contraction, which also ameliorates nitrergic (68.90±0.21 vs. 51.07±0.63, 8Hz, AngII-infused mice treated vs. non-treated). Decreased endothelial NOS expression, reduced NOS activity, and augmented levels of TNF-α, and ROS were found following AngII-infusion. These alterations were prevented, or at least decreased by anti-TLR4 antibody treatment. SIGNIFICANCE: Inhibition of TLR4 ameliorates AngII-impaired cavernosal relaxation, decreases TNF-α levels, and restores NO bioavailability, demonstrating that TLR4 partly mediates AngII-induced cavernosal dysfunction.

Obesity-induced vascular inflammation involves elevated arginase activity.

Obesity-induced vascular dysfunction involves pathological remodeling of the visceral adipose tissue (VAT) and increased inflammation. Our previous studies showed that arginase 1 (A1) in endothelial cells (ECs) is critically involved in obesity-induced vascular dysfunction. We tested the hypothesis that EC-A1 activity also drives obesity-related VAT remodeling and inflammation. Our studies utilized wild-type and EC-A1 knockout (KO) mice made obese by high-fat/high-sucrose (HFHS) diet. HFHS diet induced increases in body weight, fasting blood glucose, and VAT expansion. This was accompanied by increased arginase activity and A1 expression in vascular ECs and increased expression of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-10 (IL-10), vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein in both VAT and ECs. HFHS also markedly increased circulating inflammatory monocytes and VAT infiltration by inflammatory macrophages, while reducing reparative macrophages. Additionally, adipocyte size and fibrosis increased and capillary density decreased in VAT. These effects of HFHS, except for weight gain and hyperglycemia, were prevented or reduced in mice lacking EC-A1 or treated with the arginase inhibitor 2-(S)-amino-6-boronohexanoic acid (ABH). In mouse aortic ECs, exposure to high glucose (25 mM) and Na palmitate (200 µM) reduced nitric oxide production and increased A1, TNF-α, VCAM-1, ICAM-1, and MCP-1 mRNA, and monocyte adhesion. Knockout of EC-A1 or ABH prevented these effects. HFHS diet-induced VAT inflammation is mediated by EC-A1 expression/activity. Limiting arginase activity is a possible therapeutic means of controlling obesity-induced vascular and VAT inflammation.

Deregulation of arginase induces bone complications in high-fat/high-sucrose diet diabetic mouse model.

A balanced diet is crucial for healthy development and prevention of musculoskeletal related diseases. Diets high in fat content are known to cause obesity, diabetes and a number of other disease states. Our group and others have previously reported that activity of the urea cycle enzyme arginase is involved in diabetes-induced dysregulation of vascular function due to decreases in nitric oxide formation. We hypothesized that diabetes may also elevate arginase activity in bone and bone marrow, which could lead to bone-related complications. To test this we determined the effects of diabetes on expression and activity of arginase, in bone and bone marrow stromal cells (BMSCs). We demonstrated that arginase 1 is abundantly present in the bone and BMSCs. We also demonstrated that arginase activity and expression in bone and bone marrow is up-regulated in models of diabetes induced by HFHS diet and streptozotocin (STZ). HFHS diet down-regulated expression of healthy bone metabolism markers (BMP2, COL-1, ALP, and RUNX2) and reduced bone mineral density, bone volume and trabecular thickness. However, treatment with an arginase inhibitor (ABH) prevented these bone-related complications of diabetes. In-vitro study of BMSCs showed that high glucose treatment increased arginase activity and decreased nitric oxide production. These effects were reversed by treatment with an arginase inhibitor (ABH). Our study provides evidence that deregulation of l-arginine metabolism plays a vital role in HFHS diet-induced diabetic complications and that these complications can be prevented by treatment with arginase inhibitors. The modulation of l-arginine metabolism in disease could offer a novel therapeutic approach for osteoporosis and other musculoskeletal related diseases.

Arginase inhibition enhances angiogenesis in endothelial cells exposed to hypoxia.

Hypoxia-induced arginase elevation plays an essential role in several vascular diseases but influence of arginase on hypoxia-mediated angiogenesis is completely unknown. In this study, in vitro network formation in bovine aortic endothelial cells (BAEC) was examined after exposure to hypoxia for 24h with or without arginase inhibition. Arginase activity, protein levels of the two arginase isoforms, eNOS, and VEGF as well as production of NO and ROS were examined to determine the involvement of arginase in hypoxia-mediated angiogenesis. Hypoxia elevated arginase activity and arginase 2 expression but reduced active p-eNOS(Ser1177) and NO levels in BAEC. In addition, both VEGF protein levels and endothelial elongation and network formation were reduced with continued hypoxia, whereas ROS levels increased and NO levels decreased. Arginase inhibition limited ROS, restored NO formation and VEGF expression, and prevented the reduction of angiogenesis. These results suggest a fundamental role of arginase activity in regulating angiogenic function.

Requirement of NOX2 expression in both retina and bone marrow for diabetes-induced retinal vascular injury.

OBJECTIVE: Diabetic retinopathy, a major cause of blindness, is characterized by increased expression of vascular endothelial growth factor (VEGF), leukocyte attachment to the vessel walls and increased vascular permeability. Previous work has shown that reactive oxygen species (ROS) produced by the superoxide generating enzyme NOX2/NADPH oxidase play a crucial role in the vascular pathology. The aim of this work was to identify the cellular sources of the damaging NOX2 activity by studies using bone marrow chimera mice. METHODS: Bone marrow cells were collected from the femurs and tibias of wild type and NOX2 deficient (NOX2(-/-)) donor mice and injected intravenously into lethally irradiated NOX2(-/-) and wild type recipients. Following recovery from radiation, mice were rendered diabetic by streptozotocin injections. The following groups of bone marrow chimeras were studied: non-diabetic WT → WT, diabetic WT → WT, diabetic WT → NOX2(-/-), diabetic NOX2(-/-) → WT. After 4 weeks of diabetes, early signs of retinopathy were examined by measuring ROS, expression of VEGF and ICAM-1, leukocyte attachment to the vessel wall and vascular permeability. RESULTS: The retinas of the diabetic WT → WT chimeras showed significant increases in ROS as compared with the non-diabetic chimeras. These diabetes-induced alterations were correlated with increases in expression of VEGF and ICAM-1, leukocyte adhesion and vascular permeability. Each of these diabetes-induced alterations were significantly attenuated in the diabetic WT → NOX2(-/-) and NOX2(-/-) → WT chimera groups (p<0.05). CONCLUSION: NOX2-generated ROS produced by both bone marrow-derived cells and resident retinal cells contribute importantly to retinal vascular injury in the diabetic retina. Targeting NOX2 in bone marrow and/or retinal cells may represent a novel therapeutic strategy for the treatment/prevention of vascular injury in the diabetic retina.

Akita spontaneously type 1 diabetic mice exhibit elevated vascular arginase and impaired vascular endothelial and nitrergic function.

BACKGROUND: Elevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice. METHODS AND RESULTS: Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser(1177) (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks. CONCLUSIONS: Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.

Superoxide anion production by NADPH oxidase plays a major role in erectile dysfunction in middle-aged rats: prevention by antioxidant therapy.

INTRODUCTION.: Prevalence of erectile dysfunction (ED) increases progressively with aging, but the ED pathophysiology at its early stages is still poorly investigated. AIM.: This study aimed to evaluate the functional and molecular alterations of erectile function at middle age, focusing on the contribution of oxidative stress in erectile tissue for the ED. METHODS.: Young (3.5-month) and middle-aged (10-month) male Wistar rats were used. Rat corpus cavernosum (RCC) was dissected free and mounted in 10-mL organ baths containing Krebs solution. Intracavernosal pressure (ICP) in anesthetized rats was evaluated. MAIN OUTCOME MEASURES.: Concentration-response curves to endothelium-dependent and endothelium-independent agents, as well as to electrical field stimulation (EFS), were obtained in RCC strips. Measurement of cyclic guanosine monophosphate (cGMP) and expressions of neuronal nitric oxide synthase (nNOS) and endothelial NOS (eNOS), gp91(phox) and superoxide dismutase-1 (SOD-1) expressions in RCC were evaluated. RESULTS.: ICP was significantly reduced in middle-aged compared with young rats. RCC relaxations to acetylcholine (10(-8) to 10(-2) M), sodium nitroprusside (10(-8) to 10(-2) M), sildenafil (10(-9) to 10(-5) M), BAY 41-2272 (10(-9) to 10(-5) M), and EFS (4-32 Hz) were decreased in middle-aged group, which were nearly normalized by apocynin (NADPH oxidase inhibitor; 10(-4) M) or SOD (75 U/mL). Prolonged treatment with apocynin (85 mg/rat/day, 4 weeks) also restored the impaired relaxations in middle-aged rats. Relaxations to 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (8-Br-cGMP; 10(-8) to 3 × 10(-4) M) remained unchanged between groups. Basal and stimulated cGMP production were lower in middle-aged group, an effect fully restored by apocynin and SOD. Protein expression of nNOS and phosphorylated eNOS (p-eNOS) (Ser-1177) reduced, whereas gp(91phox) mRNA expression increased in RCC from middle-aged rats. CONCLUSIONS.: ED in middle-aged rats is associated with decreased NO bioavailability in erectile tissue due to upregulation of NADPH oxidase subunit gp91(phox) and downregulation of nNOS/p-eNOS. Antioxidant therapies may be a good pharmacological approach to prevent ED at its early stages.

Prevention of diabetes-induced arginase activation and vascular dysfunction by Rho kinase (ROCK) knockout.

AIMS: We determined the role of the Rho kinase (ROCK) isoforms in diabetes-induced vascular endothelial dysfunction and enhancement of arginase activity and expression. METHODS AND RESULTS: Studies were performed in aortic tissues from haplo-insufficient (H-I) ROCK1 and ROCK2 mice and wild-type (WT) mice rendered diabetic with streptozotocin and in bovine aortic endothelial cells (BAECs) treated with high glucose (HG, 25 mM). Protein expression of both ROCK isoforms was substantially elevated in aortas of WT mice after 8 weeks of diabetes and in BAECs after 48 h in HG. Impairment of endothelium-dependent vasorelaxation of aortas was observed in diabetic WT mice. However, there was no impairment in aortas of diabetic ROCK1 H-I mice and less impairment in aortas of diabetic ROCK2 H-I mice, compared with non-diabetic mice. These vascular effects were associated with the prevention of diabetes-induced decrease in nitric oxide (NO) production and a rise in arginase activity/expression. Acute treatment with the arginase inhibitor, BEC, improved endothelium-dependent vasorelaxation of aortas of both diabetic WT and ROCK2, but not of ROCK1 mice. CONCLUSION: Partial deletion of either ROCK isoform, but to a greater extent ROCK1, attenuates diabetes-induced vascular endothelial dysfunction by preventing increased arginase activity and expression and reduction in NO production in type 1 diabetes. Limiting ROCK and arginase activity improves vascular function in diabetes.

Erectile function is improved in aged rats by PnTx2-6, a toxin from Phoneutria nigriventer spider venom.

INTRODUCTION: Age-associated erectile dysfunction (ED) involves a decrease in nitric oxide (NO) availability and impaired relaxation. PnTx2-6, a toxin from the Phoneutria nigriventer spider, has been demonstrated to improve erectile function via NO/cyclic guanosine monophosphate (cGMP) pathway. This spider's venom is characterized by several symptoms, including erection. PnTx2-6 has been implicated in this phenomenon. Animal venoms have been postulated as potential drugs to treat ED. AIM: PnTx2-6 toxin improves erectile function in aged rats via NO/cGMP. We investigated the effect of PnTx2-6 in the erectile function of aged rats. MAIN OUTCOME MEASURES: ED was evaluated through changes in intracavernosal pressure/mean arterial pressure ratio during electrical field stimulation (EFS) of the pelvic ganglion of aged and adult rats (70 vs. 14 weeks). In functional studies, EFS-induced relaxation of corpus cavernosum (CC) strips were performed with or without PnTx2-6 (10-8M). RESULTS: The decrease in erectile function associated with age was partially restored 15-20 minutes after injection of PnTx2-6 and further improved by sildenafil. PnTx2-6 enhanced EFS-induced relaxation, as well as cGMP levels in CC, from young and aged rats. Relaxation due to PnTx2-6 was further increased after 30 minutes incubation with Y-27632, a Rho-kinase inhibitor (10-6 M), in aging CC. Nitric oxide synthase (NOS) activity in aged and young cavernosal tissue was increased by incubation with PnTx2-6 (10 minutes). However, this toxin did not modify NOS expression. CONCLUSION: PnTx2-6 improves penile relaxation in aged rats, via increased NOS activity and NO release, resulting in enhanced cGMP levels.

Extracellular signal-regulated kinase (ERK) inhibition decreases arginase activity and improves corpora cavernosal relaxation in streptozotocin (STZ)-induced diabetic mice.

INTRODUCTION: Increased arginase activity (AA) has been implicated in hypertension and diabetes-induced endothelial dysfunction by reducing L-arginine availability and nitric oxide production. Higher levels of active extracellular signal-regulated kinase (ERK) have been found in patients with erectile dysfunction (ED) compared to patients without it. Both ERK and arginase have been reported to affect the expression and activity of nitric oxide synthase (NOS) and consequently penile erection. Nevertheless, signaling pathways activated by ERK in the penis are not well known. AIM: We hypothesized that inhibition of ERK by ERK inhibitor PD98059 decreases AA and thus improves cavernosal relaxation in streptozotocin (STZ)-diabetic mice. METHODS: The AA, ERK, eNOS, and arginase I and II expressions were examined through Western blot, and functional response of cavernosal tissue were determined. Control and diabetic cavernosal tissues were pretreated with PD98059 (10(-5) M) and arginase inhibitor ((S)-(2-boronoethyl)-L-cysteine hydrochloride, [BEC]10(-4) M]). MAIN OUTCOME MEASURES: Diabetes increased AA significantly (twofold) over control mice and this effect was blocked by acute treatment with PD98059. Cavernosal strips from diabetic mice exhibited decreased relaxation (STZ-diabetic vs. control, respectively) to both the endothelium-dependent agonist acetylcholine (38.0 ± 5% vs. 82.5 ± 7%) and nitrergic stimulation (27 ± 2% vs. 76 ± 6%) by electrical field stimulation (EFS, 1-32 Hz). However, this impairment in cavernosal relaxation from diabetic mice was attenuated by treatment with PD98059 in nitrergic (27 ± 2% vs. 60 ± 4%) and endothelium-dependent relaxation responses (38.0 ± 5% vs. 67.5 ± 6%). Acute treatment with the arginase inhibitor BEC (10(-4) M) also improves EFS-induced relaxation in diabetic mice (31 ± 3% vs. 49 ± 2%). Moreover, vascular expression of activated ERK was increased in diabetic over control mice. CONCLUSION: These data suggest that ERK inhibition prevents elevation of penile AA and protects against ED caused by diabetes.

Calpain mediates pulmonary vascular remodeling in rodent models of pulmonary hypertension, and its inhibition attenuates pathologic features of disease.

Pulmonary hypertension is a severe and progressive disease, a key feature of which is pulmonary vascular remodeling. Several growth factors, including EGF, PDGF, and TGF-ß1, are involved in pulmonary vascular remodeling during pulmonary hypertension. However, increased knowledge of the downstream signaling cascades is needed if effective clinical interventions are to be developed. In this context, calpain provides an interesting candidate therapeutic target, since it is activated by EGF and PDGF and has been reported to activate TGF-ß1. Thus, in this study, we examined the role of calpain in pulmonary vascular remodeling in two rodent models of pulmonary hypertension. These data showed that attenuated calpain activity in calpain-knockout mice or rats treated with a calpain inhibitor resulted in prevention of increased right ventricular systolic pressure, right ventricular hypertrophy, as well as collagen deposition and thickening of pulmonary arterioles in models of hypoxia- and monocrotaline-induced pulmonary hypertension. Additionally, inhibition of calpain in vitro blocked intracellular activation of TGF-ß1, which led to attenuated Smad2/3 phosphorylation and collagen synthesis. Finally, smooth muscle cells of pulmonary arterioles from patients with pulmonary arterial hypertension showed higher levels of calpain activation and intracellular active TGF-ß. Our data provide evidence that calpain mediates EGF- and PDGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells via an intracrine TGF-ß1 pathway in pulmonary hypertension.