Chronic 3D Vascular-Immune Interface Established by Coculturing Pressurized Resistance Arteries and Immune Cells.

[Figure: see text].

Toll-like receptor protein 4 monoclonal antibody inhibits mmLDL-induced endothelium-dependent vasodilation dysfunction of mouse mesenteric arteries.

Minimally modified low-density lipoprotein (mmLDL) is a risk factor for cardiovascular disease. This study was designed to investigate the effect of a Toll-like receptor 4 monoclonal antibody (TLR4 mAb) on mmLDL-induced endothelium-dependent vasodilation (EDV) impairment in mouse mesenteric arteries and to explore the underlying mechanism. Animals were divided into a normal control group, an mmLDL treatment group, and a TLR4 mAb intervention group. The serum concentrations of IL-1ß and TNF-α were detected using enzyme-linked immunosorbent assays (ELISAs). EDV function was measured using a microvascular tension tracing method. The protein levels and mRNA expression of IL-1ß and TNF-α in vascular tissue were detected using western blot analysis and reverse transcription polymerase chain reaction, respectively. TLR4 mAb improved mmLDL-induced EDV functional impairment in a dose-dependent manner. TLR4 mAb significantly upregulated KCa3.1 and KCa2.3 channel protein levels and downregulated TNF-α and IL-1ß expression. These effects were possibly associated with the competitive antagonism of TLR4 mAb on the TLR4 signaling pathway and the downstream NF-κB p65 and p38 MAPK pathways, which are activated by mmLDL. In conclusion, pretreatment with TLR4 mAb lessens mmLDL-induced EDV dysfunction and inhibits overexpression of inflammatory factors. Regulation of the TLR4 pathway, as well as its downstream NF-κB p65 and p38 MAPK pathways, may be an effective strategy for the prevention and treatment of cardiovascular diseases.

PM2.5 upregulates rat mesenteric arteries 5-HT2A receptor via inflammatory-mediated mitogen-activated protein kinases signaling pathway.

Fine particulate matter (PM2.5 ) is an important environmental risk factor for cardiovascular diseases. However, little is known about the effects of PM2.5 on arteries. The present study investigated whether PM2.5 alters 5-hydroxytryptamine (5-HT) receptor expression and inflammatory mediators on rat mesenteric arteries, and examined the underlying mechanisms. Isolated rat mesenteric arteries segments were cultured with PM2.5 in the presence or absence of ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was monitored by a sensitive myograph. The expression of 5-HT2A/1B receptors and inflammatory mediators were studied by a real-time polymerase chain reaction and/or by immunohistochemistry. The phosphorylation of mitogen-activated protein kinases (MAPK) pathway was detected by Western blot. Compared with the fresh or culture alone groups, 1.0 µg/mL PM2.5 cultured for 16 hours significantly enhanced contractile response induced by 5-HT and increased 5-HT2A receptor mRNA and protein expressions, indicating PM2.5 upregulates 5-HT2A receptor. SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly decreased PM2.5 -induced elevated contraction and mRNA and protein expression of 5-HT2A receptor. Cultured with PM2.5 significantly increased the mRNA expression of inflammatory mediators (NOS2, IL-1ß, and TNF-α), while SB203580 decreased mRNA expression level of NOS2, IL-1ß, and TNF-α. SP600125 (JNK inhibitor) decreased mRNA expression level of TNF-α and IL-1ß. After PM2.5 exposure, the phosphorylation of p38 and ERK1/2 protein were increased. SB203580 and U0126 inhibited the PM2.5 caused increased phosphorylation protein of p38 and ERK1/2. In conclusion, PM2.5 induces inflammatory-mediated MAPK pathway in artery which subsequently results in enhanced vascular contraction responding to 5-HT via the upregulated 5-HT2A receptors.

Schistosome Egg Migration: Mechanisms, Pathogenesis and Host Immune Responses.

Many parasitic worms possess complex and intriguing life cycles, and schistosomes are no exception. To exit the human body and progress to their successive snail host, Schistosoma mansoni eggs must migrate from the mesenteric vessels, across the intestinal wall and into the feces. This process is complex and not always successful. A vast proportion of eggs fail to leave their definite host, instead becoming lodged within intestinal or hepatic tissue, where they can evoke potentially life-threatening pathology. Thus, to maximize the likelihood of successful egg passage whilst minimizing host pathology, intriguing egg exit strategies have evolved. Notably, schistosomes actively exert counter-inflammatory influences on the host immune system, discreetly compromise endothelial and epithelial barriers, and modulate granuloma formation around transiting eggs, which is instrumental to their migration. In this review, we discuss new developments in our understanding of schistosome egg migration, with an emphasis on S. mansoni and the intestine, and outline the host-parasite interactions that are thought to make this process possible. In addition, we explore the potential immune implications of egg penetration and discuss the long-term consequences for the host of unsuccessful egg transit, such as fibrosis, co-infection and cancer development.

Reactive oxygen species derived from NAD(P)H oxidase play a role on ethanol-induced hypertension and endothelial dysfunction in rat resistance arteries.

Chronic ethanol consumption is a risk factor for cardiovascular diseases. We studied whether NAD(P)H oxidase-derived reactive oxygen species (ROS) play a role in ethanol-induced hypertension, vascular dysfunction, and protein expression in resistance arteries. Male Wistar rats were treated with ethanol (20 % v/v) for 6 weeks. Ethanol treatment increased blood pressure and decreased acetylcholine-induced relaxation in the rat mesenteric arterial bed (MAB). These responses were attenuated by apocynin (30 mg/kg/day; p.o. gavage). Ethanol consumption increased superoxide anion (O2-) generation and decreased nitrate/nitrite (NO x ) concentration in the rat MAB and apocynin prevented these responses. Conversely, ethanol did not affect the concentration of hydrogen peroxide (H2O2) and reduced glutathione (GSH) or the activity of superoxide dismutase (SOD) and catalase (CAT) in the rat MAB. Ethanol increased interleukin (IL)-10 levels in the rat MAB but did not affect the levels of tumor necrosis factor (TNF)-α, IL-6, or IL-1ß. Ethanol increased the expression of Nox2 and the phosphorylation of SAPK/JNK, but reduced eNOS expression in the rat MAB. Apocynin prevented these responses. However, ethanol treatment did not affect the expression of Nox1, Nox4, p38MAPK, ERK1/2, or SAPK/JNK in the rat MAB. Ethanol increased plasma levels of TBARS, TNF-α, IL-6, IL-1ß, and IL-10, whereas it decreased NO x levels. The major finding of our study is that NAD(P)H oxidase-derived ROS play a role on ethanol-induced hypertension and endothelial dysfunction in resistance arteries. Moreover, ethanol consumption affects the expression and phosphorylation of proteins that regulate vascular function and NAD(P)H oxidase-derived ROS play a role in such responses.

Lipopolysaccharides, but not Angiotensin ll, lnduces Direct Pro-lnflammatory Effects in Cultured Mouse Arteries and Human Endothelial and Vascular Smooth Muscle Cells.

Angiotensin II (Ang II) might induce pro-inflammatory effects directly in the vascular wall independently of its haemodynamic effects. The aim of our study was to investigate the putative direct pro-inflammatory and vasomotor effects of Ang II and compare to those of lipopolysaccharides (LPS) in mouse isolated mesenteric resistance-sized arteries (MRA) supported by experiments in cultured human primary endothelial and vascular smooth muscle cells. Results showed that 24-hr organ culture of mouse MRA with 10 nM Ang II had, unlike 100 ng/mL LPS, no effects on IL-6 or MCP-1 secretion, VCAM1 mRNA expression or endothelial function, while Ang II significantly decreased maximal vasomotor responses to phenylephrine. In support, 24-hr organ culture of mouse MRA significantly suppressed Agtr1a mRNA and augmented Tlr4 mRNA along with attenuated vasomotor responses to Ang II. Moreover, contrary to LPS and TNF-α, Ang II and [Sar1]-Ang II had no concentration- or time-dependent effects on IL-6 and MCP-1 secretion in human umbilical vein endothelial cells (HUVEC) and human aortic smooth muscle cells (HASMC). AGTR1 or AGTR2 mRNA expression was undetectable in HUVEC, whereas HASMC expressed only AGTR1 mRNA. In summary, contrary to previous studies and the observed effects of LPS, we could not demonstrate direct vascular pro-inflammatory effects of Ang II ex vivo or in vitro. As indicated by our results, down-regulation or desensitization of AT1 R during culture may explain our findings.

Effects of long-term active immunization with the second extracellular loop of human ß1- or ß3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats.

OBJECTIVE: To evaluate whether active immunization producing ß1- or ß3-antibodies (ß1-ABs and ß3-ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA). DESIGN AND METHOD: Lewis rats were immunized for 6months with peptidic sequences corresponding to the second extracellular loop of ß1- and ß3-adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various ß-AR agonists, phenylephrine and KCl. RESULTS: The immunizations producing functional ß1-ABs and ß3-ABs did not affect the SBP. However, in TA from ß1-AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with ß3-AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with ß1- or ß3-peptides, relaxations induced by the various ß-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. CONCLUSIONS: Our study shows that ß1- and ß3-ABs can affect vascular reactivity. ß1-ABs would have a pathogenic action whereas ß3-ABs would have a beneficial effect on aorta reactivity.

The angiotensin II type 2 receptor activates flow-mediated outward remodelling through T cells-dependent interleukin-17 production.

AIMS: The angiotensin II type 1 receptor (AT1R) through the activation of immune cells plays a key role in arterial inward remodelling and reduced blood flow in cardiovascular disorders. On the other side, flow (shear stress)-mediated outward remodelling (FMR), involved in collateral arteries growth in ischaemic diseases, allows revascularization. We hypothesized that the type 2 receptor (AT2R), described as opposing the effects of AT1R, could be involved in FMR. METHODS AND RESULTS: We studied FMR using a model of ligation of feed arteries supplying collateral pathways in the mouse mesenteric arterial bed in vivo. Seven days after ligation, diameter increased by 30% in high flow (HF) arteries compared with normal flow vessels. FMR was absent in mice lacking AT2R. At Day 2, T lymphocytes expressing AT2R were present preferentially around HF arteries. FMR did not occur in athymic (nude) mice lacking T cells and in mice treated with anti-CD3ε antibodies. AT2R activation induced interleukin-17 production by memory T cells. Treatment of nude mice or AT2R-deficient mice with interleukin-17 restored diameter enlargement in HF arteries. Interleukin-17 increased NO-dependent relaxation and matrix metalloproteinases activity, both important in FMR. Remodelling of feeding arteries in the skin flap model of ischaemia was also absent in AT2R-deficient mice and in anti-interleukin-17-treated mice. Finally, remodelling, absent in 12-month-old mice, was restored by a treatment with the AT2R non-peptidic agonist C21. CONCLUSION: AT2R-dependent interleukin-17 production by T lymphocyte is necessary for collateral artery growth and could represent a new therapeutic target in ischaemic disorders.

Arterial stiffness is associated with adipokine dysregulation in non-hypertensive obese mice.

The aim of this study was to characterize alterations in vascular structure and mechanics in murine mesenteric arteries from obese non-hypertensive mice, as well as their relationship with adipokines. Four-week old C57BL/6J male mice were assigned either to a control (C, 10% kcal from fat) or a high-fat diet (HFD, 45% kcal from fat) for 32weeks. HFD animals weighed 30% more than controls (p<0.001), exhibited similar blood pressure, increased leptin, insulin and superoxide anion (O2(-)) levels, and reduced adiponectin levels and nitric oxide (NO) bioavailability. Arterial structure showed an outward remodeling with an increase in total number of both adventitial and smooth muscle cells in HFD. Moreover, HFD mice exhibited an increased arterial stiffness assessed by ß-values (C=2.4±0.5 vs HFD=5.3±0.8; p<0.05) and aortic pulse wave velocity (PWV, C=3.4±0.1 vs HFD=3.9±0.1; p<0.05). ß-Values and PWV positively correlated with leptin, insulin or O2(-) levels, whereas they negatively correlated with adiponectin levels and NO bioavailability (p<0.01). A reduction in fenestrae number together with an increase in type-I collagen amount (p<0.05) were observed in HFD. These data demonstrate that HFD accounts for the development of vascular remodeling and arterial stiffness associated with adipokine dysregulation and oxidative stress, independently of hypertension development.

Macrophage depletion lowers blood pressure and restores sympathetic nerve α2-adrenergic receptor function in mesenteric arteries of DOCA-salt hypertensive rats.

We tested the hypothesis that vascular macrophage infiltration and O2 (-) release impairs sympathetic nerve α2-adrenergic autoreceptor (α2AR) function in mesenteric arteries (MAs) of DOCA-salt hypertensive rats. Male rats were uninephrectomized or sham operated (sham). DOCA pellets were implanted subcutaneously in uninephrectomized rats who were provided high-salt drinking water or high-salt water with apocynin. Sham rats received tap water. Blood pressure was measured using radiotelemetry. Treatment of sham and DOCA-salt rats with liposome-encapsulated clodronate was used to deplete macrophages. After 3-5, 10-13, and 18-21 days of DOCA-salt treatment, MAs and peritoneal fluid were harvested from euthanized rats. Norepinephrine (NE) release from periarterial sympathetic nerves was measured in vitro using amperometry with microelectrodes. Macrophage infiltration into MAs as well as TNF-α and p22(phox) were measured using immunohistochemistry. Peritoneal macrophage activation was measured by flow cytometry. O2 (-) was measured using dihydroethidium staining. Hypertension developed over 28 days, and apocynin reduced blood pressure on days 18-21. O2 (-) and macrophage infiltration were greater in DOCA-salt MAs compared with sham MAs after day 10. Peritoneal macrophage activation occurred after day 10 in DOCA-salt rats. Macrophages expressing TNF-α and p22(phox) were localized near sympathetic nerves. Impaired α2AR function and increased NE release from sympathetic nerves occurred in MAs from DOCA-salt rats after day 18. Macrophage depletion reduced blood pressure and vascular O2 (-) while restoring α2AR function in DOCA-salt rats. Macrophage infiltration into the vascular adventitia contributes to increased blood pressure in DOCA-salt rats by releasing O2 (-), which disrupts α2AR function, causing enhanced NE release from sympathetic nerves.

Prenatal lipopolysaccharide exposure causes mesenteric vascular dysfunction through the nitric oxide and cyclic guanosine monophosphate pathway in offspring.

Cardiovascular diseases, such as hypertension, could be programmed in fetal life. Prenatal lipopolysaccharide (LPS) exposure in utero results in increased blood pressure in offspring, but the vascular mechanisms involved are unclear. Pregnant Sprague-Dawley rats were intraperitoneally injected with LPS (0.79mg/kg) or saline (0.5ml) on gestation days 8, 10, and 12. The offspring of LPS-treated dams had higher blood pressure and decreased acetylcholine (ACh)-induced relaxation and increased phenylephrine (PE)-induced contraction in endothelium-intact mesenteric arteries. Endothelium removal significantly enhanced the PE-induced contraction in offspring of control but not LPS-treated dams. The arteries pretreated with l-NAME to inhibit nitric oxide synthase (eNOS) in the endothelium or ODQ to inhibit cGMP production in the vascular smooth muscle had attenuated ACh-induced relaxation but augmented PE-induced contraction to a larger extent in arteries from offspring of control than those from LPS-treated dams. In addition, the endothelium-independent relaxation caused by sodium nitroprusside was also decreased in arteries from offspring of LPS-treated dams. The functional results were accompanied by a reduction in the expressions of eNOS and soluble guanylate cyclase (sGC) and production of NO and cGMP in arteries from offspring of LPS-treated dams. Furthermore, LPS-treated dam's offspring arteries had increased oxidative stress and decreased antioxidant capacity. Three-week treatment with TEMPOL, a reactive oxygen species (ROS) scavenger, normalized the alterations in the levels of ROS, eNOS, and sGC, as well as in the production of NO and cGMP and vascular function in the arteries of the offspring of LPS-treated dams. In conclusion, prenatal LPS exposure programs vascular dysfunction of mesenteric arteries through increased oxidative stress and impaired NO-cGMP signaling pathway.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats.

AIMS: Hypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. MAIN METHODS: SHR and Wistar rats were treated with anti-TLR4 antibody (1µg/day) or unspecific IgG for 15days (i.p.). KEY FINDINGS: Anti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SIGNIFICANCE: Together, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Endothelial dysfunction and altered mechanical and structural properties of resistance arteries in a murine model of graft-versus-host disease.

A putative involvement of the vasculature seems to play a critical role in the pathophysiology of graft-versus-host disease (GVHD). We aimed to characterize alterations of mesenteric resistance arteries in GVHD in a fully MHC-mismatched model of BALB/c mice conditioned with total body irradiation that underwent transplantation with bone marrow cells and splenocytes from syngeneic (BALB/c) or allogeneic (C57BL/6) donors. After 4 weeks, animals were sacrificed and mesenteric resistance arteries were studied in a pressurized myograph. The expression of endothelial (eNOS) and inducible nitric oxide (NO)-synthase (iNOS) was quantified and vessel wall ultrastructure was investigated with electron microscopy. The myograph study revealed an endothelial dysfunction in allogeneic-transplant recipients, whereas endothelium-independent vasodilation was similar to syngeneic-transplant recipients or untreated controls. The expression of eNOS was decreased and iNOS increased, possibly contributing to endothelial dysfunction. Additionally, arteries of allogeneic transplant recipients exhibited a geometry-independent increase in vessels strain. For both findings, electron microscopy provided a structural correlate by showing severe damage of the whole vessel wall in allogeneic-transplant recipient animals. Our study provides further data to prove, and is the first to characterize, functional and structural vascular alterations in the early course after allogeneic transplantation directly in an ex vivo setting and, therefore, strongly supports the hypothesis of a vascular form of GVHD.

The CXCR1/2 ligand NAP-2 promotes directed intravascular leukocyte migration through platelet thrombi.

Thrombosis promotes leukocyte infiltration into inflamed tissues, leading to organ injury in a broad range of diseases; however, the mechanisms by which thrombi guide leukocytes to sites of vascular injury remain ill-defined. Using mouse models of endothelial injury (traumatic or ischemia reperfusion), we demonstrate a distinct process of leukocyte recruitment, termed "directed intravascular migration," specifically mediated by platelet thrombi. Single adherent platelets and platelet aggregates stimulated leukocyte shape change at sites of endothelial injury; however, only thrombi were capable of inducing directed intravascular leukocyte migration. Leukocyte recruitment and migration induced by platelet thrombi occurred most prominently in veins but could also occur in arteries following ischemia-reperfusion injury. In vitro studies demonstrated a major role for platelet-derived NAP-2 (CXCL-7) and its CXCR1/2 receptor in regulating leukocyte polarization and motility. In vivo studies demonstrated the presence of an NAP-2 chemotactic gradient within the thrombus body. Pharmacologic blockade of CXCR1/2 as well as genetic deletion of NAP-2 markedly reduced leukocyte shape change and intrathrombus migration. These studies define a distinct process of leukocyte migration that is initiated by homotypic adhesive interactions between platelets, leading to the development of an NAP-2 chemotactic gradient within the thrombus body that guides leukocytes to sites of vascular injury.

Impact of local endothelial challenge with cytomegalovirus or glycoprotein B on vasodilation in intact pressurized arteries from nonpregnant and pregnant mice.

Cytomegalovirus (CMV) infections are associated with vascular diseases in the human population. We have previously shown vascular dysfunction in systemic and uterine arteries dissected from nonpregnant (NP) mouse CMV (mCMV)-infected mice that was further impaired during late pregnancy (LP). CMV attachment alone through glycoprotein B (GB) can generate signals that impact vascular tone regulation. However, the contribution of direct virus interactions with endothelium to the vascular dysfunction we previously observed after in vivo mCMV infection is not known. We used a pressure myograph system to infuse GB or whole intact mCMV inside arteries dissected from uninfected mice and assessed vasodilation to methacholine infused inside pressurized arteries rather than applied abluminally. These results were compared to those observed after methacholine infusion into untreated arteries dissected from mCMV-infected mice. In mesenteric arteries, vasodilation to infused methacholine did not differ among treatments in NP or LP groups in contrast to previously published studies. However, increased vasoconstrictor activity was unmasked after blocking thromboxane receptors or prostaglandin production. Vasodilation in uterine arteries from uninfected NP mice to infused methacholine was increased by both GB and whole intact mCMV pretreatment. Untreated uterine arteries from mCMV-infected NP mice showed even greater vasodilation. There was no effect of GB or whole intact mCMV pretreatment in uterine arteries from uninfected LP mice, whereas vasodilation to infused methacholine was reduced in untreated uterine arteries from mCMV-infected LP mice. CMV exerts direct effects on vascular function which should be considered during viral reactivation leading to viremia and during GB-based vaccine administration.

Interferon-gamma induced adipose tissue inflammation is linked to endothelial dysfunction in type 2 diabetic mice.

Interferon-gamma (IFNγ) has previously been associated with immuno-mediated inflammation in diet-induced obesity and type 1 diabetes. This study sought to define the role of IFNγ-induced adipose tissue inflammation in endothelial dysfunction in type 2 diabetes. We examined mesenteric adipose tissue (MAT) inflammation, and endothelial function of small mesenteric artery (SMA) in control mice (m Lepr(db)), diabetic mice (Lepr(db)), m Lepr(db) treated with IFNγ, and Lepr(db) treated with anti-IFNγ or anti-monocyte chemoattractant protein-1 (anti-MCP-1). mRNA and protein expression of IFNγ and MCP-1 were increased in MAT of Lepr(db), accompanied by increased T-lymphocyte and macrophage infiltration. Anti-IFNγ reduced MAT inflammatory cell infiltration and inflammatory cytokine expression in Lepr(db), while IFNγ treatment showed the opposite effects in m Lepr(db). Acetylcholine (ACh)-induced vasorelaxation of SMA was impaired in Lepr(db) versus m Lepr(db), but sodium nitroprusside (SNP)-induced vasorelaxation was comparable. Both anti-IFNγ and anti-MCP-1 improved endothelial function of Lepr(db), while IFNγ treatment impaired endothelial function of m Lepr(db). Superoxide production was higher in both MAT and SMA of Lepr(db) mice, and anti-IFNγ reduced MAT and SMA superoxide production. Macrophage accumulation in the adventitia of SMA, and mRNA expression of MCP-1 in SMA were increased in Lepr(db) and IFNγ-treated m Lepr(db), but reduced in anti-IFNγ treated Lepr(db). These findings suggest IFNγ has a key role in the regulation of visceral adipose tissue inflammatory response and endothelial dysfunction in type 2 diabetes.

In vitro protection of vascular function from oxidative stress and inflammation by pulsatility in resistance arteries.

OBJECTIVE: Resistance arteries remain subject to pulsatility, a potent regulator of large elastic artery tone and structure, but the effect is incompletely understood. Extracorporeal circulation during cardiac surgery is often associated with absence of pulsatility, which may affect vascular tone. To define the role of the vascular wall in the inflammatory process that may occur with or without pulsatility, we studied resistance arteries functions ex vivo. We measured vascular reactivity, oxidative stress, and inflammation in the arterial wall. METHODS: Isolated rat mesenteric resistance arteries were mounted in an arteriograph and subjected to pulsatility or not in vitro. Arteries were perfused with a physiologic salt solution without circulating cells. RESULTS: After 180 minutes, flow-mediated dilation was higher and pressure-induced myogenic tone lower in arteries subjected to pulsatility. Without pulsatility, reactive oxygen species and markers of inflammation (monocyte chemotactic protein 1 and tumor necrosis factor α) were higher than baseline. In perfused mesenteric beds under similar conditions, tumor necrosis factor α was higher in perfusate after 180 minutes of nonpulsatility (5.7 ± 1.6 pg/mL vs 1.1 ± 0.4 pg/mL; P < .01). In arteries treated with the antioxidant 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (tempol), flow-mediated dilation and myogenic tone were similar in nonpulsatile and pulsatile arteries; monocyte chemotactic protein 1 and nuclear factor κB expression levels were not increased in tempol-treated nonpulsatile arteries. CONCLUSIONS: Absence of pulsatility in resistance arteries increased oxidative stress, which in turn induced inflammation and preferentially altered pressure and flow-dependent tone, which play a key role in control of local blood flow.

Innate immunity, through late complement components activation, contributes to the development of early vascular inflammation and morphologic alterations in experimental diabetes.

OBJECTIVE: To verify if innate immunity, and namely the assembly of terminal complement complex (TCC) could be involved in the development of early diabetic vascular damage. METHODS AND RESULTS: At first in 2 groups of diabetic or non-diabetic Wistar rats the occurrence of basal or stimulated stable adherence to the endothelial layer and extravasation of circulating fluorescently-labelled leukocytes was assessed by using an in vivo videomicroscopy technique. In a second part of the study, the development of vascular damage in short term diabetes was studied in the genetically C6 deficient rats of the PVG strain, and compared with those observed in the wild-type C6 sufficient animals. Here, the analysis of mesentery vascular expression of mRNA for vascular cell adhesion molecule (VCAM)-1, transforming growth factor-ß (TGF-ß), connective tissue growth factor (CTGF), and platelet-derived growth factor (PDGF), the evaluation of intravascular protein levels of VCAM-1, TGF-ß, CTGF, proliferative cell nuclear antigen (PCNA), as well as the assessment of structural changes and Complement components deposition at the mesentery arterial vascular wall were also performed. CONCLUSIONS: Leukocyte trafficking, mesentery arteries hypertrophy, extracellular matrix deposition, local vascular gene and protein expression of VCAM-1, TGF-ß, CTGF and PCNA, as well as PGDF gene expression were all increased by short term diabetes, but all significantly reduced in the C6 deficient diabetic animals, thus suggesting an active role for TCC in the development of vascular inflammation in the early phases of experimental diabetes.

A novel role for calpain in the endothelial dysfunction induced by activation of angiotensin II type 1 receptor signaling.

RATIONALE: The cytosolic protease calpain has been recently implicated in the vascular remodeling of angiotensin II (Ang II) type 1 receptor (AT(1)R) signaling. The role of Ang II/AT(1)R/calpain signaling on endothelial function, an important and early determinant of vascular pathology, remains though totally unknown. Accordingly, we investigated the role of calpain in the endothelial dysfunction of Ang II. OBJECTIVE: To demonstrate a mechanistic role for calpain in the endothelial dysfunction induced by Ang II/AT(1)R signaling. To establish endothelial-expressed calpains as an important target of AT(1)R signaling. METHODS AND RESULTS: Subchronic administration of nonpressor doses of Ang II to rats and mice significantly increased vascular calpain activity via AT(1)R signaling. Intravital microscopy studies revealed that activation of vascular expressed calpains causes endothelial dysfunction with increased leukocyte-endothelium interactions and albumin permeability in the microcirculation. Western blot and immunohistochemistry studies confirmed that Ang II/AT(1)R signaling preferentially activates the constitutively expressed µ-calpain isoform and demonstrated a calpain-dependent degradation of IκBα, along with upregulation of nuclear factor κB-regulated endothelial cell adhesion molecules. These physiological and biochemical parameters were nearly normalized following inhibition of AT(1)R or calpain in vivo. RNA silencing studies in microvascular endothelial cells, along with knockout and transgenic mouse studies, further confirmed the role of µ-calpain in the endothelial adhesiveness induced by Ang II. CONCLUSIONS: This study uncovers a novel role for calpain in the endothelial dysfunction of Ang II/AT(1)R signaling and establishes the calpain system as a novel molecular target of the vascular protective action of renin-angiotensin system inhibition. Our results may have significant clinical implications in vascular disease.

T regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury.

Angiotensin (Ang) II induces hypertension by mechanisms mediated in part by adaptive immunity and T effector lymphocytes. T regulatory lymphocytes (Tregs) suppress T effector lymphocytes. We questioned whether Treg adoptive transfer would blunt Ang II-induced hypertension and vascular injury. Ten- to 12-week-old male C57BL/6 mice were injected IV with 3 ×10(5) Treg (CD4(+)CD25(+)) or T effector (CD4(+)CD25(-)) cells, 3 times at 2-week intervals, and then infused or not with Ang II (1 µg/kg per minute, SC) for 14 days. Ang II increased systolic blood pressure by 43 mm Hg (P<0.05), NADPH oxidase activity 1.5-fold in aorta and 1.8-fold in the heart (P<0.05), impaired acetylcholine vasodilatory responses by 70% compared with control (P<0.05), and increased vascular stiffness (P<0.001), mesenteric artery vascular cell adhesion molecule expression (2-fold; P<0.05), and aortic macrophage and T-cell infiltration (P<0.001). All of the above were prevented by Treg but not T effector adoptive transfer. Ang II caused a 43% decrease in Foxp3(+) cells in the renal cortex, whereas Treg adoptive transfer increased Foxp3(+) cells 2-fold compared with control. Thus, Tregs suppress Ang II-mediated vascular injury in part through anti-inflammatory actions. Immune mechanisms modulate Ang II-induced blood pressure elevation, vascular oxidative stress, inflammation, and endothelial dysfunction.