Systemic Administration of Pegylated Arginase-1 Attenuates the Progression of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a serious complication of diabetes that results from sustained hyperglycemia, hyperlipidemia, and oxidative stress. Under these conditions, inducible nitric oxide synthase (iNOS) expression is upregulated in the macrophages (MΦ) and microglia, resulting in increased production of reactive oxygen species (ROS) and inflammatory cytokines, which contribute to disease progression. Arginase 1 (Arg1) is a ureohydrolase that competes with iNOS for their common substrate, L-arginine. We hypothesized that the administration of a stable form of Arg1 would deplete L-arginine's availability for iNOS, thus decreasing inflammation and oxidative stress in the retina. Using an obese Type 2 diabetic (T2DM) db/db mouse, this study characterized DR in this model and determined if systemic treatment with pegylated Arg1 (PEG-Arg1) altered the progression of DR. PEG-Arg1 treatment of db/db mice thrice weekly for two weeks improved visual function compared with untreated db/db controls. Retinal expression of inflammatory factors (iNOS, IL-1ß, TNF-α, IL-6) was significantly increased in the untreated db/db mice compared with the lean littermate controls. The increased retinal inflammatory and oxidative stress markers in db/db mice were suppressed with PEG-Arg1 treatment. Additionally, PEG-Arg1 treatment restored the blood-retinal barrier (BRB) function, as evidenced by the decreased tissue albumin extravasation and an improved endothelial ZO-1 tight junction integrity compared with untreated db/db mice.

Protection against Doxorubicin-Induced Cardiotoxicity through Modulating iNOS/ARG 2 Balance by Electroacupuncture at PC6.

BACKGROUND: Doxorubicin (DOX) is a commonly used chemotherapeutic drug but is limited in clinical applications by its cardiotoxicity. Neiguan acupoint (PC6) is a well-recognized acupoint for the treatment of cardiothoracic disease. However, whether acupuncture at PC6 could be effective in preventing DOX-induced cardiotoxicity is still unknown. METHODS: A set of experiments were performed with myocardial cells, wild type, inducible nitric oxide synthase knockout (iNOS-/-), and myocardial-specific ablation arginase 2 (Myh6-ARG 2-/-) mice. We investigated the protective effect and the underlying mechanisms for electroacupuncture (EA) against DOX-induced cardiotoxicity by echocardiography, immunostaining, biochemical analysis, and molecular biotechnology in vivo and in vitro analysis. RESULTS: We found that DOX-mediated nitric oxide (NO) production was positively correlated with the iNOS level but has a negative correlation with the arginase 2 (ARG 2) level in both myocardial cells and tissues. Meanwhile, EA at PC6 alleviated cardiac dysfunction and cardiac hypertrophy in DOX-treated mice. EA at PC6 blocked the upregulation of NO production in accompanied with the downregulated iNOS and upregulated ARG 2 levels in myocardial tissue induced by DOX. Furthermore, knockout iNOS prevented cardiotoxicity and EA treatment did not cause the further improvement of cardiac function in iNOS-/- mice treated by DOX. In contrast, deficiency of myocardial ARG 2 aggravated DOX-induced cardiotoxicity and reduced EA protective effect. CONCLUSION: These results suggest that EA treatment at PC6 can prevent DOX-induced cardiotoxicity through modulating NO production by modulating the iNOS/ARG 2 balance in myocardial cells.

Utility of LysM-cre and Cdh5-cre Driver Mice in Retinal and Brain Research: An Imaging Study Using tdTomato Reporter Mouse.

Purpose: The lysozyme 2 (Lyz2 or LysM) cre mouse is extensively used to achieve genetic manipulation in myeloid cells and it has been widely employed in retinal research. However, LysM has been recently described to be expressed in brain neurons and there is a debate on whether it is also expressed by resident microglia in addition to infiltrating macrophages. Methods: We examined LysM-cre recombination in retinal tissue using a LysM-cre/tdTomato reporter mouse together with immunolabeling for several retinal cell markers. We further compared LysM-cre tdTomato recombination with that of Cdh5-cre driver, which is expressed in both endothelial and hematopoietic cells. Results: LysM-cre was strongly expressed in most microglia/resident macrophages in neonatal retinas (P8) and to a lesser extent in microglia of adult retinas. In addition, there was some neuronal recombination (8 %) of LysM-cre specifically in adult retinal ganglion cells and amacrine cells. After retinal ischemia-reperfusion injury, LysM-cre was strongly expressed in microglia/infiltrating macrophages. Cdh5-cre was expressed in endothelial and myeloid cells of P8 pups retinas. Unexpectedly, Cdh5 showed additional expression in adult mouse retinal ganglion cells and brain neurons. Conclusions: LysM-cre is expressed in macrophages and a subset of microglia together with a small but significant recombination of LysM-cre in the retinal neurons of adult mice. Cdh5 also showed some neuronal expression in both retina and brain of adult mice. These findings should be taken into consideration when interpreting results from central nervous system research using LysM-cre and Cdh5-cre mice.

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.

Mechanisms of Diabetes-Induced Endothelial Cell Senescence: Role of Arginase 1.

We have recently found that diabetes-induced premature senescence of retinal endothelial cells is accompanied by NOX2-NADPH oxidase-induced increases in the ureohydrolase enzyme arginase 1 (A1). Here, we used genetic strategies to determine the specific involvement of A1 in diabetes-induced endothelial cell senescence. We used A1 knockout mice and wild type mice that were rendered diabetic with streptozotocin and retinal endothelial cells (ECs) exposed to high glucose or transduced with adenovirus to overexpress A1 for these experiments. ABH [2(S)-Amino-6-boronohexanoic acid] was used to inhibit arginase activity. We used Western blotting, immunolabeling, quantitative PCR, and senescence associated β-galactosidase (SA β-Gal) activity to evaluate senescence. Analyses of retinal tissue extracts from diabetic mice showed significant increases in mRNA expression of the senescence-related proteins p16INK4a, p21, and p53 when compared with non-diabetic mice. SA β-Gal activity and p16INK4a immunoreactivity were also increased in retinal vessels from diabetic mice. A1 gene deletion or pharmacological inhibition protected against the induction of premature senescence. A1 overexpression or high glucose treatment increased SA β-Gal activity in cultured ECs. These results demonstrate that A1 is critically involved in diabetes-induced senescence of retinal ECs. Inhibition of arginase activity may therefore be an effective therapeutic strategy to alleviate diabetic retinopathy by preventing premature senescence.

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.

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.

Activation of the endothelin system mediates pathological angiogenesis during ischemic retinopathy.

Retinopathy of prematurity adversely affects premature infants because of oxygen-induced damage of the immature retinal vasculature, resulting in pathological neovascularization (NV). Our pilot studies using the mouse model of oxygen-induced retinopathy (OIR) showed marked increases in angiogenic mediators, including endothelins and endothelin receptor (EDNR) A. We hypothesized that activation of the endothelin system via EDNRA plays a causal role in pathological angiogenesis and up-regulation of angiogenic mediators, including vascular endothelial growth factor A (VEGFA) in OIR. Mice were exposed to 75% oxygen from post-natal day P7 to P12, treated with either vehicle or EDNRA antagonist BQ-123 or EDNRB antagonist BQ-788 on P12, and kept at room air from P12 to P17 (ischemic phase). RT-PCR analysis revealed increased levels of EDN2 and EDNRA mRNA, and Western blot analysis revealed increased EDN2 expression during the ischemic phase. EDNRA inhibition significantly increased vessel sprouting, resulting in enhanced physiological angiogenesis and decreased pathological NV, whereas EDNRB inhibition modestly improved vascular repair. OIR triggered significant increases in VEGFA protein and mRNA for delta-like ligand 4, apelin, angiopoietin-2, and monocyte chemoattractant protein-1. BQ-123 treatment significantly reduced these alterations. EDN2 expression was localized to retinal glia and pathological NV tufts of the OIR retinas. EDN2 also induced VEGFA protein expression in cultured astrocytes. In conclusion, inhibition of the EDNRA during OIR suppresses pathological NV and promotes physiological angiogenesis.

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.

Arginase as a mediator of diabetic retinopathy.

We have shown previously that diabetes causes increases in retinal arginase activity that are associated with impairment of endothelial cell (EC)-dependent vasodilation and increased formation of the peroxynitrite biomarker nitrotyrosine. Arginase blockade normalizes vasodilation responses and reduces nitrotyrosine formation, suggesting that overactive arginase contributes to diabetic retinopathy by reducing NO and increasing oxidative stress. We tested this hypothesis by studies in streptozotocin-induced diabetic mice and high glucose (HG) treated retinal ECs. Our results show that arginase activity is increased in both diabetic retinas and HG-treated retinal ECs as compared with the controls. Western blot shows that both arginase isoforms are present in retinal vessels and ECs and arginase I is increased in the diabetic vessels and HG-treated retinal ECs. Nitrate/nitrite levels are significantly increased in diabetic retinas, indicating an increase in total NO products. However, levels of nitrite, an indicator of bioavailable NO, are reduced by diabetes. Imaging analysis of NO formation in retinal sections confirmed decreases in NO formation in diabetic retinas. The decrease in NO is accompanied by increased [Formula: see text] formation and increased leukocyte attachment in retinal vessels. Studies in knockout mice show that arginase gene deletion enhances NO formation, reduces [Formula: see text] and prevents leukostasis in the diabetic retinas. HG treatment of retinal ECs also reduces NO release, increases oxidative stress, increases ICAM-1, and induces EC death. Arginase inhibitor treatment reverses these effects. In conclusion, diabetes- and HG-induced signs of retinal vascular activation and injury are associated with increased arginase activity and expression, decreased bioavailable NO, and increased [Formula: see text] formation. Blockade of the arginase pathway prevents these alterations, suggesting a primary role of arginase in the pathophysiological process.

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.

Toxicity and cellular uptake of gold nanorods in vascular endothelium and smooth muscles of isolated rat blood vessel: importance of surface modification.

Gold nanorods (GNRs) have promising applications in drug delivery and cancer treatment and are generally administered via direct injection into the circulation. Thus it is necessary to evaluate their potential adverse effects on blood vessels. Herein, GNRs with various surface modifications are used to evaluate the toxicity and cellular uptake of GNRs into vascular endothelial and smooth muscle cells of isolated rat aortic rings. Surfactant-capped GNRs are synthesized and either coated with polyelectrolyte (PE) to prepare PE-GNRs, or modified with thiolated polyethylene glycol (PEG) to prepare PEG-GNRs. Using toxicity assays, small-vessel myography, fluorescence microscopy, and electron microscopy, it is shown that therapeutic concentrations of PE-GNRs but not PEG-GNRs are toxic to the vascular endothelium, which leads to an impaired relaxation function of aortic rings. However, no toxicity to smooth muscles is observed. Moreover, electron microscopy analysis confirms the cellular uptake of PE-GNRs but not PEG-GNRs into the endothelium of exposed aortic rings. The difference in toxicity and cellular uptake of PE-GNRs versus PEG-GNRs is explained and linked to free surfactant molecules and protein adsorption, respectively. The results indicate that toxicity and cellular uptake in the vascular endothelium in blood vessels are potential adverse effects of systemically administered GNR solutions, which can be prevented by appropriate surface functionalization.

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.

Tissue-intrinsic dysfunction of circadian clock confers transplant arteriosclerosis.

The suprachiasmatic nucleus of the brain is the circadian center, relaying rhythmic environmental and behavioral information to peripheral tissues to control circadian physiology. As such, central clock dysfunction can alter systemic homeostasis to consequently impair peripheral physiology in a manner that is secondary to circadian malfunction. To determine the impact of circadian clock function in organ transplantation and dissect the influence of intrinsic tissue clocks versus extrinsic clocks, we implemented a blood vessel grafting approach to surgically assemble a chimeric mouse that was part wild-type (WT) and part circadian clock mutant. Arterial isografts from donor WT mice that had been anastamosed to common carotid arteries of recipient WT mice (WT:WT) exhibited no pathology in this syngeneic transplant strategy. Similarly, when WT grafts were anastamosed to mice with disrupted circadian clocks, the structural features of the WT grafts immersed in the milieu of circadian malfunction were normal and absent of lesions, comparable to WT:WT grafts. In contrast, aortic grafts from Bmal1 knockout (KO) or Period-2,3 double-KO mice transplanted into littermate control WT mice developed robust arteriosclerotic disease. These lesions observed in donor grafts of Bmal1-KO were associated with up-regulation in T-cell receptors, macrophages, and infiltrating cells in the vascular grafts, but were independent of hemodynamics and B and T cell-mediated immunity. These data demonstrate the significance of intrinsic tissue clocks as an autonomous influence in experimental models of arteriosclerotic disease, which may have implications with regard to the influence of circadian clock function in organ transplantation.

Angiotensin II-induced vascular endothelial dysfunction through RhoA/Rho kinase/p38 mitogen-activated protein kinase/arginase pathway.

Enhanced vascular arginase activity impairs endothelium-dependent vasorelaxation by decreasing l-arginine availability to endothelial nitric oxide (NO) synthase, thereby reducing NO production. Elevated angiotensin II (ANG II) is a key component of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated arginase activity. We determined signaling mechanisms by which ANG II increases endothelial arginase function. Results show that ANG II (0.1 µM, 24 h) elevates arginase activity and arginase I expression in bovine aortic endothelial cells (BAECs) and decreases NO production. These effects are prevented by the arginase inhibitor BEC (100 µM). Blockade of ANG II AT(1) receptors or transfection with small interfering RNA (siRNA) for Gα12 and Gα13 also prevents ANG II-induced elevation of arginase activity, but siRNA for Gαq does not. ANG II also elevates active RhoA levels and induces phosphorylation of p38 MAPK. Inhibitors of RhoA activation (simvastatin, 0.1 µM) or Rho kinase (ROCK) (Y-27632, 10 µM; H1152, 0.5 µM) block both ANG II-induced elevation of arginase activity and phosphorylation of p38 MAPK. Furthermore, pretreatment of BAECs with p38 inhibitor SB-202190 (2 µM) or transfection with p38 MAPK siRNA prevents ANG II-induced increased arginase activity/expression and maintains NO production. Additionally, inhibitors of p38 MAPK (SB-203580, 5 µg·kg(-1)·day(-1)) or arginase (ABH, 8 mg·kg(-1)·day(-1)) or arginase gene knockout in mice prevents ANG II-induced vascular endothelial dysfunction and associated enhancement of arginase. These results indicate that ANG II increases endothelial arginase activity/expression through Gα12/13 G proteins coupled to AT(1) receptors and subsequent activation of RhoA/ROCK/p38 MAPK pathways leading to endothelial dysfunction.