Noureddine Idris-Khodja

Dual opposing roles of adaptive immunity in hypertension

Hypertension involves remodelling and inflammation of the arterial wall. Interactions between vascular and inflammatory cells play a critical role in disease initiation and progression. T effector and regulatory lymphocytes, members of the adaptive immune system, play contrasting roles in hypertension. Signals from the central nervous system and the innate immune system antigen-presenting cells activate T effector lymphocytes and promote their differentiation towards pro-inflammatory T helper (Th) 1 and Th17 phenotypes. Th1 and Th17 effector cells, via production of pro-inflammatory mediators, participate in the low-grade inflammation that leads to blood pressure elevation and end-organ damage. T regulatory lymphocytes, on the other hand, counteract hypertensive effects by suppressing innate and adaptive immune responses. The present review summarizes and discusses the adaptive immune mechanisms that participate in the pathophysiology in hypertension.

Endothelin-1 Overexpression Exaggerates Diabetes-Induced Endothelial Dysfunction by Altering Oxidative Stress.

BACKGROUND Increased endothelin (ET)-1 expression causes endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we hypothesized that overexpression of ET-1 in the endothelium would exaggerate diabetes-induced endothelial dysfunction. METHODS Diabetes was induced by streptozotocin treatment (55mg/kg/day, i.p.) for 5 days in 6-week-old male wild type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Small mesenteric artery (MA) endothelial function and vascular remodeling by pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription/quantitative PCR were determined. RESULTS Endothelium-dependent vasodilatory responses to acetylcholine of MA were reduced 24% by diabetes in WT ( P < 0.05), and further decreased by 12% in eET-1 ( P < 0.05). Diabetes decreased MA media/lumen in WT and eET-1 ( P < 0.05), whereas ET-1 overexpression increased MA media/lumen similarly in diabetic and nondiabetic WT mice ( P < 0.05). Vascular ROS production was increased 2-fold by diabetes in WT ( P < 0.05) and further augmented 1.7-fold in eET-1 ( P < 0.05). Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3 ) expression in eET-1 by 31% ( P < 0.05) but not in WT. Induction of diabetes caused a 52% ( P < 0.05) increase in superoxide dismutase 1 ( Sod1 ) and a 32% ( P < 0.05) increase in Sod2 expression in WT but not in eET-1. CONCLUSIONS Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by decrease in eNOS expression, increase in vascular oxidative stress, and decrease in antioxidant capacity.

Matrix metalloproteinase-2 knockout prevents angiotensin II-induced vascular injury

Aims Matrix metalloproteinases (MMPs) have been implicated in the development of hypertension in animal models and humans. Mmp2 deletion did not change Ang II-induced blood pressure (BP) rise. However, whether Mmp2 knockout affects angiotensin (Ang) II-induced vascular injury has not been tested. We sought to determine whether Mmp2 knockout will prevent Ang II-induced vascular injury. Methods and results A fourteen-day Ang II infusion (1000 ng/kg/min, SC) increased systolic BP, decreased vasodilatory responses to acetylcholine, induced mesenteric artery (MA) hypertrophic remodelling, and enhanced MA stiffness in wild-type (WT) mice. Ang II enhanced aortic media and perivascular reactive oxygen species generation, aortic vascular cell adhesion molecule-1 and monocyte chemotactic protein-1 expression, perivascular monocyte/macrophage and T cell infiltration, and the fraction of spleen activated CD4+CD69+ and CD8+CD69+ T cells, and Ly-6Chi monocytes. Study of intracellular signalling showed that Ang II increased phosphorylation of epidermal growth factor receptor and extracellular-signal-regulated kinase 1/2 in vascular smooth muscle cells isolated from WT mice. All these effects were reduced or prevented by Mmp2 knockout, except for systolic BP elevation. Ang II increased Mmp2 expression in immune cells infiltrating the aorta and perivascular fat. Bone marrow (BM) transplantation experiments revealed that in absence of MMP2 in immune cells, Ang II-induced BP elevation was decreased, and that when MMP2 was deficient in either immune or vascular cells, Ang II-induced endothelial dysfunction was blunted. Conclusions Mmp2 knockout impaired Ang II-induced vascular injury but not BP elevation. BM transplantation revealed a role for immune cells in Ang II-induced BP elevation, and for both vascular and immune cell MMP2 in Ang II-induced endothelial dysfunction.

Selective ROS-dependent p53-associated anticancer effects of the hypoxoside derivative rooperol on human teratocarcinomal cancer stem-like cells

Cancer stem cells (CSCs) are potential targets for innovative anticancer therapies that involve natural products with potential chemopreventive effects. We therefore analyzed the antineoplastic activity of rooperol, the aglycone of the plant-derived compound hypoxoside, on a model of Oct4-expressing cancer stem-like cell, i.e. the human embryonal carcinoma (EC) cell NT2/D1.Rooperol selectively inhibited the proliferation of NT2/D1 cells in a concentration-dependent manner and had no effect on either normal embryonic fibroblasts which are more restrictive pluripotent stem cells or on NCCIT p53-mutant EC cells. Accordingly, rooperol only eliminates colon carcinoma cells expressing p53.Rooperol treatment triggered cell death on NT2/D1 cells through the alteration of mitochondrial membrane potential and production of reactive oxygen species (ROS). Rooperol-induced apoptosis was associated with activation of p53 and concentration-dependent changes of the expression levels of both caspase 3 and poly ADP ribose polymerase type 1 cleaved subunits. These modifications were accompanied by a downregulation of Oct4 and its two partners involved in the maintenance of cell pluripotency and self-renewal, Nanog and Sox2.Treatment with intracellular membrane permeant O2− scavengers prevented rooperol-induced apoptosis and upregulation of the expression of p53 and active caspase-3. Our findings indicate that rooperol mediates its growth inhibitory effects on CSCs via a mitochondrial redox-sensitive mechanism. We propose that abrogating the expression of the stemness regulators is a prerequisite for rooperol to fully exert its pro-apoptotic properties on wild-type p53-bearing CSCs.

OS 21-01 NOX1 OR NOX4 DELETION PREVENTS TYPE 1 DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION.

Objective : The prognosis of type-1 diabetes remains poor and is primarily related to the increased risk of vascular complications. Overproduction of reactive oxygen species by NADPH oxidase (NOX) is believed to play an important role in diabetes-related vascular injury. NOX1 may play a role in the macrovascular disease, whereas NOX4 may have protective actions. Nevertheless, their role in diabetic microangiopathy is less well understood. We hypothesized that deletion of Nox1 would prevent diabetes-induced endothelial dysfunction and vascular remodeling of small arteries whereas Nox4 would exaggerate vascular injury. Design and Method : Diabetes-related vascular injury were studied in atherosclerosis-prone apolipoprotein knockout (Apoe-/-) mice. Six-week-old male Apoe-/- mice, Apoe-/- mice deficient in Nox1 (Apoe-/-/Nox1y/-) and Nox4 (Apoe-/-/Nox4-/-) were rendered diabetic by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days. Mice were studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Results: Apoe-/- mice presented a maximal endothelium-dependent vasodilatory response to acetylcholine of only 46%, which was further decreased by ∼50% by diabetes. In contrast, endothelium-dependent relaxations to acetylcholine were 1.5-fold higher in diabetic Apoe-/-/Nox1y/- and Apoe-/-/Nox4-/- mice compared to vehicle-treated Apoe-/- mice. Endothelium-independent relaxation responses to the nitric oxide donor, sodium nitroprusside, were similar in all groups. Diabetes decreased MA stiffness in Apoe-/- mice, as indicated by a rightward displacement of the stress-strain curves, which was blunted by Nox1 or Nox4 knockout. MA media/lumen was unaltered by diabetes. Knockout of Nox4 but not Nox1 increased MA media/lumen 1.4-fold in diabetic Apoe-/- mice. Conclusions: These results suggest that NOX1 and NOX4 play a pathophysiological role in diabetes-induced endothelial dysfunction and contribute to potentially maladaptive changes in vascular stiffness. NOX4 also seems to have dual actions on the vasculature, as it is also protective against vascular remodeling of small arteries in type 1 diabetes.

MPS 18-02 ENDOTHELIN-1 OVEREXPRESSION PRESERVES ENDOTHELIAL FUNCTION IN MICE WITH VASCULAR SMOOTH MUSCLE CELL-SPECIFIC DELETION OF PPAR-GAMMA

Objective: Peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;) agonists reduce blood pressure (BP) and vascular injury in hypertensive rodents. Ppar&ggr; inactivation in vascular smooth muscle cells (VSMC) using a tamoxifen inducible Cre-Lox system enhanced angiotensin II-induced vascular damage. Transgenic mice overexpressing endothelin (ET)-1 in the endothelium (eET-1) exhibit endothelial dysfunction, increased oxidative stress and inflammation. We hypothesized that inactivation of the Ppar gene in VSMC (smPpar&ggr;−/−) will exaggerate ET-1-induced vascular damage. Design and Method: Eleven week-old male control, eET-1, smPpar&ggr;−/− and eET-1/smPpar&ggr;−/− mice were treated with tamoxifen (1 mg/kg/day, s.c.) for 5 days and sacrificed 4 weeks later. BP was measured by telemetry. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining, monocyte/macrophage infiltration by immunofluorescence and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA) or perivascular fat (PVAT). Spleen T cell and monocyte profiles were assessed by flow cytometry. Results: Systolic BP was 20 mmHg higher in eET-1 and unaffected by Ppar&ggr; inactivation. MA vasorelaxation to acetylcholine was impaired 37% only in smPpar&ggr;−/−. Likewise, ET-1-induced contractions were enhanced only in smPpar&ggr;−/−. ROS levels were increased 1.7-fold in smPpar&ggr;−/− and 2.5-fold in eET-1/smPpar&ggr;−/−. Monocyte/macrophage infiltration in PVAT was 2-fold higher in eET-1 and smPpar&ggr;−/−, which was further increased 2-fold in eET-1/smPpar&ggr;−/−. The percentage of CD11b+ cells was increased 2.3-fold in smPpar&ggr;−/− and further increased 1.5-fold in eET-1/smPpar&ggr;−/−. The percentage of Ly-6Chi monocytes was increased ∼1.8-fold in eET-1 and smPpar&ggr;−/− but not eET-1/smPpar&ggr;−/−. The percentage of T regulatory cells was increased 1.5-fold in smPpar&ggr;−/− and decreased by 26% in eET-1, which was further decreased by 38% in eET-1/smPpar&ggr;−/−. Conclusions: These results suggest that increased ET-1 paradoxically preserves endothelial function in mice with inactivated VSMC Ppar&ggr; despite enhanced oxidative stress. Flow cytometry data indicate that infiltrating monocyte/macrophages in these mice might be anti-inflammatory.

PS 16-31 ENDOTHELIN-1 EXAGGERATES TYPE-1 DIABETES-ACCELERATED ATHEROSCLEROSIS THROUGH NADPH OXIDASES 1 AND 4

Objective: NADPH oxidase (NOX) 1 but not NOX4-dependent oxidative stress plays a role in diabetic vascular disease, including atherosclerosis. Endothelin (ET)-1 has been implicated in diabetes-induced vascular complications. We showed that crossing mice overexpressing ET-1 selectively in endothelium (eET-1) with apolipoprotein E knockout (Apoe−/−) mice exaggerated high-fat diet-induced atherosclerosis in part by increasing oxidative stress. We hypothesized that ET-1 overexpression in the endothelium would exaggerate diabetes-accelerated atherosclerosis through a mechanism involving NOX1 but not NOX4. Design and method: Six-week-old male Apoe−/− mice, eET-1/Apoe−/− and eET-1/Apoe−/− mice deficient in Nox1 (eET-1/Apoe−/−/Nox1y/−) or Nox4 (eET-1/Apoe−/−/Nox4−/−) were rendered diabetic with 55 mg/kg/day streptozotocin (STZ) IP for 5 days and studied 14 weeks later. Endothelial function and vascular remodeling were assessed in mesenteric arteries (MA) using pressurized myography. Aortic atherosclerotic lesions were quantified using Oil Red O staining. Results: Diabetic Apoe−/− mice presented an impaired maximal endothelium-dependent vasodilatory response to acetylcholine (21%), which was not observed in diabetic eET-1/Apoe−/−, eET-1/Apoe−/−/Nox1y/− or eET-1/Apoe−/−/Nox4−/− mice (99%). Endothelium-independent relaxation to the nitric oxide (NO) donor sodium nitroprusside was similar between groups. Blockade of NO synthase with L-NAME completely blunted endothelium-dependent relaxation to acetylcholine in diabetic eET-1/Apoe−/− mice, which was prevented by Nox1 but not by Nox4 knockout. ET-1 overexpression caused a 1.8-fold increase in MA media/lumen of diabetic Apoe−/− mice, which was further exaggerated 1.2-fold by Nox4 but not Nox1 knockout. ET-1 overexpression exaggerated > 2-fold the atherosclerotic lesion area in the aortic sinus in diabetic Apoe−/− mice, which was reduced ∼40% by Nox1 and Nox4 knockout. Conclusions: Increased levels of ET-1 exaggerate diabetes-accelerated atherosclerosis through NOX1 and NOX4, despite paradoxically improving endothelium-dependent relaxation in small arteries.

LBPS 01-03 ENDOTHELIN-1 OVEREXPRESSION EXAGGERATES DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION BY ALTERING OXIDATIVE STRESS BALANCE

Objective: Increased endothelin (ET)-1 expression has been shown to cause endothelial dysfunction and oxidative stress. Plasma ET-1 is increased in patients with diabetes mellitus. Since endothelial dysfunction often precedes vascular complications in diabetes, we sought to determine whether ET-1 contributes to diabetes-induced endothelial dysfunction. We hypothesized that overexpression of ET-1 in the endothelium will exaggerate diabetes-induced endothelial dysfunction. Design and Method: Diabetes was induced by streptozotocin treatment (STZ, 55 mg/kg/day, ip) for 5 days in 6 week-old male wild-type (WT) mice and in mice overexpressing human ET-1 restricted to the endothelium (eET-1). Mice were studied 14 weeks later. Endothelial function and vascular remodeling using pressurized myography, reactive oxygen species (ROS) production by dihydroethidium staining and mRNA expression by reverse transcription-quantitative PCR were assessed in mesenteric arteries (MA). Results: MA endothelium-dependent vasodilatory responses to acetylcholine were reduced 24% by diabetes in WT, and further decreased by 12% in eET-1. Diabetes decreased MA media/lumen in WT and eET-1 whereas ET-1 overexpression increased MA media/lumen to a similar extent in diabetic and non-diabetic WT mice. Vascular ROS production in MA was increased 2-fold by diabetes in WT and further augmented 1.7-fold in eET-1. Diabetes reduced endothelial nitric oxide synthase (eNOS, Nos3) mRNA expression in eET-1 by 31% but not in WT. Induction of diabetes caused a 52% increase in superoxide dismutase 1 (Sod1) and a 32% increase in Sod2 (32%) mRNA expression in WT but not in eET-1. Conclusions: Increased expression of ET-1 exaggerates diabetes-induced endothelial dysfunction. This may be caused by a decrease in eNOS expression, an increase in vascular oxidative stress and a decrease in antioxidant capacity.

MPS 18-08 MAPPING OF CHROMOSOME 2 REGIONS LINKED TO VASCULAR INFLAMMATION USING CONGENIC RATS

Objective: Chromosome 2 (chr2) introgression from normotensive Brown Norway rats (BN) into hypertensive Dahl salt sensitive (SS) background (consomic SS2BN) reduced blood pressure (BP) and vascular inflammation under a normal-salt diet. Mapping of chr2 using congenic rats revealed that the distal portion of BN chr2 (SS2BNa) but not the middle segment (SS2BNb) on the SS background under a normal-salt diet contains anti-inflammatory genes. However, the role of chr2 in the regulation of vascular inflammation under a high-salt diet (HSD) remains unknown. We hypothesized that SS2BNa but not SS2BNb rats would have reduced vascular inflammation under HSD. Design and Method: Four-to-6 week old male SS, SS2BNa and SS2BNb rats were fed a HSD (4% NaCl) for 8 weeks or until they developed a stroke as manifested by seizures. Vascular remodeling was assessed in mesenteric arteries (MA) and middle cerebral arteries (MCA) using pressurized myography. Reactive oxygen species (ROS) production by dihydroethidium staining, vascular cell adhesion molecule (VCAM)-1 expression and CD3+ T cell infiltration by immunofluorescence were determined in aorta or perivascular adipose tissue (PVAT). BP was measured by telemetry after 6 weeks of HSD. Results: Systolic BP tended to be higher in SS2BNb compared to SS. The incidence of seizures was 2.5-fold higher in SS2BNb. MA media/lumen was 1.3-fold higher in SS2BNa (P < 0.01). MCA stiffness was increased in SS2BNa, as indicated by a leftward displacement of the stress-strain curve. PVAT ROS production was 1.8-fold higher in SS2BNa (P < 0.01) and tended to be lower in SS2BNb. Aortic VCAM-1 was increased 2.1-fold in SS2BNa (P < 0.05). Aortic PVAT CD3+ T cell infiltration was 55% lower in SS2BNb (P < 0.05). Conclusions: Unexpectedly, SS2BNa rats present increased vascular injury under HSD. The absence of vascular inflammation and remodeling in SS2BNb rats despite slightly higher BP seems maladaptive and may explain the increased incidence of stroke.