Safia Ogbi

Adaptive Cerebral Neovascularization in a Model of Type 2 Diabetes: Relevance to Focal Cerebral Ischemia

OBJECTIVE The effect of diabetes on neovascularization varies between different organ systems. While excessive angiogenesis complicates diabetic retinopathy, impaired neovascularization contributes to coronary and peripheral complications of diabetes. However, how diabetes influences cerebral neovascularization is not clear. Our aim was to determine diabetes-mediated changes in the cerebrovasculature and its impact on the short-term outcome of cerebral ischemia. RESEARCH DESIGN AND METHODS Angiogenesis (capillary density) and arteriogenesis (number of collaterals and intratree anostomoses) were determined as indexes of neovascularization in the brain of control and type 2 diabetic Goto-Kakizaki (GK) rats. The infarct volume, edema, hemorrhagic transformation, and short-term neurological outcome were assessed after permanent middle–cerebral artery occlusion (MCAO). RESULTS The number of collaterals between middle and anterior cerebral arteries, the anastomoses within middle–cerebral artery trees, the vessel density, and the level of brain-derived neurotrophic factor were increased in diabetes. Cerebrovascular permeability, matrix metalloproteinase (MMP)-9 protein level, and total MMP activity were augmented while occludin was decreased in isolated cerebrovessels of the GK group. Following permanent MCAO, infarct size was smaller, edema was greater, and there was no macroscopic hemorrhagic transformation in GK rats. CONCLUSIONS The augmented neovascularization in the GK model includes both angiogenesis and arteriogenesis. While adaptive arteriogenesis of the pial vessels and angiogenesis at the capillary level may contribute to smaller infarction, changes in the tight junction proteins may lead to the greater edema following cerebral ischemia in diabetes.

Cytochrome c oxidase subunit IV as a marker of protein kinase Cε function in neonatal cardiac myocytes: implications for cytochrome c oxidase activity

We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) alpha and epsilon from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCalpha or epsilon activation. Intact cell phorbol ester treatment caused a decline in the in vitro (32)P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca(2+) and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated (32)P in particulate fractions isolated from 4beta-PMA-treated cells in a Ca(2+)-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCepsilon-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962-24966] into NCMs before 4beta-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4beta-PMA-treated NCMs, the PKCepsilon isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCepsilon function in NCMs. Furthermore, PKCepsilon probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.

Circulating mitochondrial DNA and Toll-like receptor 9 are associated with vascular dysfunction in spontaneously hypertensive rats

AIMS Immune system activation is a common feature of hypertension pathogenesis. However, the mechanisms that initiate this activation are not well understood. Innate immune system recognition and response to danger are becoming apparent in many cardiovascular diseases. Danger signals can arise from not only pathogens, but also damage-associated molecular patterns (DAMPs). Our first hypothesis was that the DAMP, mitochondrial DNA (mtDNA), which is recognized by Toll-like receptor 9 (TLR9), is elevated in the circulation of spontaneously hypertensive rats (SHR), and that the deoxyribonuclease enzymes responsible for its degradation have decreased activity in SHR. Based on these novel SHR phenotypes, we further hypothesized that (i) treatment of SHR with an inhibitory oligodinucleotide for TLR9 (ODN2088) would lower blood pressure and that (ii) treatment of normotensive rats with a TLR9-specific CpG oligonucleotide (ODN2395) would cause endothelial dysfunction and increase blood pressure. METHODS AND RESULTS We observed that SHR have elevated circulating mtDNA and diminished deoxyribonuclease I and II activity. Additionally, treatment of SHR with ODN2088 lowered systolic blood pressure. On the other hand, treatment of normotensive rats with ODN2395 increased systolic blood pressure and rendered their arteries less sensitive to acetylcholine-induced relaxation and more sensitive to norepinephrine-induced contraction. This dysfunctional vasoreactivity was due to increased cyclooxygenase and p38 mitogen-activated protein kinase activation, increased reactive oxygen species generation, and reduced nitric oxide bioavailability. CONCLUSION Circulating mtDNA and impaired deoxyribonuclease activity may lead to the activation of the innate immune system, via TLR9, and contribute to elevated arterial pressure and vascular dysfunction in SHR.

Vascular protection in diabetic stroke: role of matrix metalloprotease-dependent vascular remodeling

Temporary focal ischemia causes greater hemorrhagic transformation (HT) in diabetic Goto-Kakizaki (GK) rats, a model with increased cerebrovascular matrix metalloprotease (MMP) activity and tortuosity. The objective of the current study was to test the hypotheses that (1) diabetes-induced cerebrovascular remodeling is MMP dependent and (2) prevention of vascular remodeling by glucose control or MMP inhibition reduces HT in diabetic stroke. Control and GK rats were treated with vehicle, metformin, or minocycline for 4 weeks, and indices of remodeling including vascular tortuosity index, lumen diameter, number of collaterals, and middle cerebral artery (MCA) MMP activity were measured. Additional animals were subjected to 3 hours MCA occlusion/21 hours reperfusion, and infarct size and HT were evaluated as indices of neurovascular injury. All remodeling markers including MMP-9 activity were increased in diabetes. Infarct size was smaller in minocycline-treated animals. Both metformin and minocycline reduced vascular remodeling and severity of HT in diabetes. These results provide evidence that diabetes-mediated stimulation of MMP-9 activity promotes cerebrovascular remodeling, which contributes to greater HT in diabetes. Metformin and minocycline offer vascular protection, which has important clinical implications for diabetes patients who are at a fourfold to sixfold higher risk for stroke.

Sex-independent neuroprotection with minocycline after experimental thromboembolic stroke

BackgroundMinocycline provides neurovascular protection reducing acute cerebral injury. However, it is unclear whether minocycline is effective in females. We tested minocycline in both sexes and aged animals using a novel embolic stroke model in mice that closely mimics acute thromboembolic stroke in humans.MethodsFive groups of mice were subjected to thromboembolic stroke: adult males, aged males, adult females, aged females, and adult ovariectomized females. They were treated with phosphate saline (vehicle) or minocycline (6 mg/kg) immediately after stroke onset. Behavioral outcomes, infarct volumes and cerebral blood flow were assessed. The effect of minocycline on expression and activity of MMP-9 was analyzed.ResultsThe model resulted in reproducible infarct in the experimental groups. As expected, adult females were significantly more resistant to cerebral ischemic injury than males. This advantage was abolished by aging and ovariectomy. Minocycline significantly reduced the infarct volume (P < 0.0001) and also improved neurologic score (P < 0.0001) in all groups. Moreover, minocycline treatment significantly reduced mortality at 24 hours post stroke (P = 0.037) for aged mice (25% versus 54%). Stroke up-regulated MMP-9 level in the brain, and acute minocycline treatment reduced its expression in both genders (P < 0.0001).ConclusionIn a thromboembolic stroke model minocycline is neuroprotective irrespective of mouse sex and age.

Cerebral Myogenic Reactivity and Blood Flow in Type 2 Diabetic Rats: Role of Peroxynitrite in Hypoxia-Mediated Loss of Myogenic Tone

Dysregulation of cerebral vascular function and, ultimately, cerebral blood flow (CBF) may contribute to complications such as stroke and cognitive decline in diabetes. We hypothesized that 1) diabetes-mediated neurovascular and myogenic dysfunction impairs CBF and 2) under hypoxic conditions, cerebral vessels from diabetic rats lose myogenic properties because of peroxynitrite (ONOO−)-mediated nitration of vascular smooth muscle (VSM) actin. Functional hyperemia, the ability of blood vessels to dilate upon neuronal stimulation, and myogenic tone of isolated middle cerebral arteries (MCAs) were assessed as indices of neurovascular and myogenic function, respectively, in 10- to 12-week control and type 2 diabetic Goto-Kakizaki rats. In addition, myogenic behavior of MCAs, nitrotyrosine (NY) levels, and VSM actin content were measured under normoxic and hypoxic [oxygen glucose deprivation (OGD)] conditions with and without the ONOO− decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl) prophyrinato iron (III), chloride (FeTPPs). The percentage of myogenic tone was higher in diabetes, and forced dilation occurred at higher pressures. Functional hyperemia was impaired. Consistent with these findings, baseline CBF was lower in diabetes. OGD reduced the percentage of myogenic tone in both groups, and FeTPPs restored it only in diabetes. OGD increased VSM NY in both groups, and although FeTPPs restored basal levels, it did not correct the reduced filamentous/globular (F/G) actin ratio. Acute alterations in VSM ONOO− levels may contribute to hypoxic myogenic dysfunction, but this cannot be solely explained by the decreased F/G actin ratio due to actin nitration, and mechanisms may differ between control and diabetic animals. Our findings also demonstrate that diabetes alters the ability of cerebral vessels to regulate CBF under basal and hypoxic conditions.

Targets of vascular protection in acute ischemic stroke differ in type 2 diabetes

Hemorrhagic transformation is an important complication of acute ischemic stroke, particularly in diabetic patients receiving thrombolytic treatment with tissue plasminogen activator, the only approved drug for the treatment of acute ischemic stroke. The objective of the present study was to determine the effects of acute manipulation of potential targets for vascular protection [i.e., NF-κB, peroxynitrite, and matrix metalloproteinases (MMPs)] on vascular injury and functional outcome in a diabetic model of cerebral ischemia. Ischemia was induced by middle cerebral artery occlusion in control and type 2 diabetic Goto-Kakizaki rats. Treatment groups received a single dose of the peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)prophyrinato iron (III), the nonspecific NF-κB inhibitor curcumin, or the broad-spectrum MMP inhibitor minocycline at reperfusion. Poststroke infarct volume, edema, hemorrhage, neurological deficits, and MMP-9 activity were evaluated. All acute treatments reduced MMP-9 and hemorrhagic transformation in diabetic groups. In addition, acute curcumin and minocycline therapy reduced edema in these animals. Improved neurological function was observed in varying degrees with treatment, as indicated by beam-walk performance, modified Bederson scores, and grip strength; however, infarct size was similar to untreated diabetic animals. In control animals, all treatments reduced MMP-9 activity, yet bleeding was not improved. Neuroprotection was only conferred by curcumin and minocycline. Uncovering the underlying mechanisms contributing to the success of acute therapy in diabetes will advance tailored stroke therapies.

Reduced vascular responses to soluble guanylyl cyclase but increased sensitivity to sildenafil in female rats with type 2 diabetes

Impaired nitric oxide (NO), soluble guanylyl cyclase (sGC), and cyclic guanosine monophosphate (cGMP) signaling (NO-sGC-cGMP) has been implicated in the pathogenesis of diabetic vascular dysfunction. Efforts to directly target this signaling have led to the development of sGC agonists that activate the heme group of sGC (stimulators) or preferentially activate sGC when the heme is oxidized (activators). In this study, we hypothesized that resistance arteries from female rats with spontaneous type 2 diabetes (Goto-Kakizaki rats, GK) would have reduced vasodilatory responses to heme-dependent sGC activation and increased responses to heme-independent sGC activation compared with control rats (Wistar). Endothelium-dependent and -independent relaxation was assessed in isolated segments from mesenteric resistance arteries (MA) mounted in a wire myograph. GK MA had reduced responses to acetylcholine (pEC50: 7.96 ± 0.06 vs. 7.66 ± 0.05, P < 0.05) and sodium nitroprusside (pEC50: 8.34 ± 0.05 vs. 7.77 ± 0.04, P < 0.05). There were no group differences in 8-bromoguanosine cGMP-induced relaxation and protein kinase G1 expression (P > 0.05). GK MA had attenuated responses to BAY 41-2272 (heme-dependent sGC stimulator; pEC50: 7.56 ± 0.05 vs. 6.93 ± 0.06, P < 0.05) and BAY 58-2667 (heme-independent sGC activator; pEC50: 10.82 ± 0.07 vs. 10.27 ± 0.08, P < 0.05) and increased sensitivity to sildenafil [phosphodiesterase 5 (PDE5) inhibitor; pEC50: 7.89 ± 0.14 vs. 8.25 ± 0.13, P < 0.05]. Isolated resistance arteries from female rats of reproductive age that spontaneously develop type 2 diabetes have increased sensitivity to PDE5 inhibition and reduced responsiveness to sGC activators and stimulators.

Autoimmune therapeutic chloroquine lowers blood pressure and improves endothelial function in spontaneously hypertensive rats

It has been suggested that hypertension results from a loss of immunological tolerance and the resulting autoimmunity may be an important underlying factor of its pathogenesis. This stems from the observations that many of the features involved in autoimmunity are also implicated in hypertension. Furthermore, the underlying presence of hypertension and cardiovascular disease are frequently observed in patients with autoimmune diseases. Antimalarial agents such as chloroquine are generally among the first line treatment options for patients with autoimmune diseases; however, whether they can improve a hypertensive phenotype in a genetic model of essential hypertension remains to be clarified. Therefore, we hypothesized that chloroquine treatment would improve endothelial function and lower blood pressure in spontaneously hypertensive rats (SHR). We treated adult SHR and Wistar-Kyoto rats (12 weeks old), as well as a group of young SHR (5 weeks old), with chloroquine (40mg/kg/day via intraperitoneal injection) for 21 days. Chloroquine lowered blood pressure in adult SHR, but did not impede the development of high blood pressure in young SHR. In isolated mesenteric resistance arteries from SHR of both ages, chloroquine treatment inhibited cyclooxygenase-dependent contraction to acetylcholine, lowered vascular and systemic generation of reactive oxygen species, and improved nitric oxide bioavailability. Overall, these data reveal the anti-hypertensive mechanisms of chloroquine in the vasculature, which may be important for lowering risk of cardiovascular disease in patients with autoimmune diseases. Furthermore, it adds to the growing body of evidence suggesting that autoimmunity underlies hypertension.

Toll-Like Receptor 9–Dependent AMPKα Activation Occurs via TAK1 and Contributes to RhoA/ROCK Signaling and Actin Polymerization in Vascular Smooth Muscle Cells

Traditionally, Toll-like receptor 9 (TLR9) signals through an MyD88-dependent cascade that results in proinflammatory gene transcription. Recently, it was reported that TLR9 also participates in a stress tolerance signaling cascade in nonimmune cells. In this noncanonical pathway, TLR9 binds to and inhibits sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2), modulating intracellular calcium handling, and subsequently resulting in the activation of 5′-AMP–activated protein kinase α (AMPKα). We have previously reported that TLR9 causes increased contraction in isolated arteries; however, the mechanisms underlying this vascular dysfunction need to be further clarified. Therefore, we hypothesized that noncanonical TLR9 signaling was also present in vascular smooth muscle cells (VSMCs) and that it mediates enhanced contractile responses through SERCA2 inhibition. To test these hypotheses, aortic microsomes, aortic VSMCs, and isolated arteries from male Sprague-Dawley rats were incubated with vehicle or TLR9 agonist (ODN2395). Despite clear AMPKα activation after treatment with ODN2395, SERCA2 activity was unaffected. Alternatively, ODN2395 caused the phosphorylation of AMPKα via transforming growth factor β–activated kinase 1 (TAK1), a kinase involved in TLR9 inflammatory signaling. Downstream, we hypothesized that that TLR9 activation of AMPKα may be important in mediating actin cytoskeleton reorganization. ODN2395 significantly increased the filamentous-to-globular actin ratio, as well as indices of RhoA/Rho-associated protein kinase (ROCK) activation, with the latter being prevented by AMPKα inhibition. In conclusion, AMPKα phosphorylation after TLR9 activation in VSMCs appears to be an extension of traditional inflammatory signaling via TAK1, as opposed to SERCA2 inhibition and the noncanonical pathway. Nonetheless, TLR9-AMPKα signaling can mediate VSMC function via RhoA/ROCK activation and actin polymerization.