Blood-brain barrier breakdown and neovascularization processes after stroke and traumatic brain injury.

PURPOSE OF REVIEW: Angiogenesis or vascular reorganization plays a role in recovery after stroke and traumatic brain injury (TBI). In this review, we have focused on two major events that occur during stroke and TBI from a vascular perspective - what is the process and time course of blood-brain barrier (BBB) breakdown? and how does the surrounding vasculature recover and facilitate repair? RECENT FINDINGS: Despite differences in the primary injury, the BBB changes overlap between stroke and TBI. Disruption of BBB involves a series of events: formation of caveolae, trans and paracellular disruption, tight junction breakdown and vascular disruption. Confounding factors that need careful assessment and standardization are the severity, duration and extent of the stroke and TBI that influences BBB disruption. Vascular repair proceeds through long-term neovascularization processes: angiogenesis, arteriogenesis and vasculogenesis. Enhancing each of these processes may impart beneficial effects in endogenous recovery. SUMMARY: Our understanding of BBB breakdown acutely after the cerebrovascular injury has come a long way; however, we lack a clear understanding of the course of BBB disruption and BBB recovery and the evolution of individual cellular events associated with BBB change. Neovascularization responses have been widely studied in stroke for their role in functional recovery but the role of vascular reorganization after TBI in recovery is much less defined.

Vascularization pattern after ischemic stroke is different in control versus diabetic rats: relevance to stroke recovery.

BACKGROUND AND PURPOSE: Pre-existing diabetes mellitus worsens brain functionality in ischemic stroke. We have previously shown that type 2 diabetic rats exhibit enhanced dysfunctional cerebral neovascularization and when these rats are subjected to cerebral ischemic reperfusion injury develop hemorrhagic transformation and greater neurological deficits. However, our knowledge of vascular and functional plasticity during the recovery phase of diabetic stroke is limited. This study tested the hypothesis that vascular repair is impaired in the poststroke period in diabetes mellitus, and this is associated with poor sensorimotor and cognitive function. We further hypothesized that glycemic control prevents impaired vascularization and improves functional outcome in diabetes mellitus. METHODS: Vascularization was assessed in the ipsilateral and contralateral hemispheres in control, diabetes mellitus and diabetes mellitus plus metformin groups 14 days after ischemic reperfusion injury, as well as in respective sham controls. Three-dimensional reconstruction of the fluorescein isothiocyanate (FITC)-stained vasculature was achieved by confocal microscopy, and stereological parameters, including vascular volume and surface area, were measured. Astrogliosis was determined by glial fibrillary acidic protein staining. The relative rates of sensorimotor recovery, cognitive decline, and spontaneous activity were assessed. RESULTS: Vascular density in the peri-infarct area was significantly reduced in diabetes mellitus, whereas there was reparative neovascularization in control rats. Astroglial swelling and reactivity were more pronounced in diabetic stroke compared with control stroke. Diabetes mellitus blunted sensorimotor recovery and also exacerbated anxiety-like symptoms and cognitive deficits. Glycemic control started after stroke partially prevented these changes. CONCLUSIONS: Diabetes mellitus impairs poststroke reparative neovascularization and impedes the recovery. Glycemic control after stroke can improve neurovascular repair and improve functional outcome.

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.

Early effects of high-fat diet on neurovascular function and focal ischemic brain injury.

Obesity is a risk factor for stroke, but the early effects of high-fat diet (HFD) on neurovascular function and ischemic stroke outcomes remain unclear. The goal of this study was to test the hypotheses that HFD beginning early in life 1) impairs neurovascular coupling, 2) causes cerebrovascular dysfunction, and 3) worsens short-term outcomes after cerebral ischemia. Functional hyperemia and parenchymal arteriole (PA) reactivity were measured in rats after 8 wk of HFD. The effect of HFD on basilar artery function after middle cerebral artery occlusion (MCAO) and associated O-GlcNAcylation were assessed. Neuronal cell death, infarct size, hemorrhagic transformation (HT) frequency/severity, and neurological deficit were evaluated after global ischemia and transient MCAO. HFD caused a 10% increase in body weight and doubled adiposity without a change in lipid profile, blood glucose, and blood pressure. Functional hyperemia and PA relaxation were decreased with HFD. Basilar arteries from stroked HFD rats were more sensitive to contractile factors, and acetylcholine-mediated relaxation was impaired. Vascular O-GlcNAcylated protein content was increased with HFD. This group also showed greater mortality rate, infarct volume, HT occurrence rate, and HT severity and poor functional outcome compared with the control diet group. These results indicate that HFD negatively affects neurovascular coupling and cerebrovascular function even in the absence of dyslipidemia. These early cerebrovascular changes may be the cause of greater cerebral injury and poor outcomes of stroke in these animals.

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.

Visualizing Pericyte Mimicry of Angiotropic Melanoma by Direct Labeling of the Angioarchitecture.

In addition to intravascular dissemination, angiotropic melanoma cells have the propensity to spread along the external surface of blood vessels in a pericytic location, or pericytic mimicry. Such continuous migration without intravasation has been termed "extravascular migratory metastasis" or EVMM. In order to visualize this mechanism of tumor propagation, we used a murine brain melanoma model utilizing green fluorescent human melanoma cells and red fluorescent lectin-tagged murine vessels. This model allows the direct microscopic visualization and mapping of the interaction of melanoma cells with the brain vasculature. In this chapter, we describe the methodology of lectin perfusion to label the entire angioarchitecture in conjunction with confocal microscopy imaging to study the pericyte mimicry of the angiotropic GFP+ melanoma cells.

Early intervention of tyrosine nitration prevents vaso-obliteration and neovascularization in ischemic retinopathy.

Diabetic retinopathy and retinopathy of prematurity are blinding disorders that follow a pathological pattern of ischemic retinopathy and affect premature infants and working-age adults. Yet, the treatment options are limited to laser photocoagulation. The goal of this study is to elucidate the molecular mechanism and examine the therapeutic effects of inhibiting tyrosine nitration on protecting early retinal vascular cell death and late neovascularization in the ischemic retinopathy model. Ischemic retinopathy was developed by exposing neonatal mice to 75% oxygen [postnatal day (p) 7-p12] followed by normoxia (21% oxygen) (p12-p17). Peroxynitrite decomposition catalyst 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTPPS) (1 mg/kg), the nitration inhibitor epicatechin (10 mg/kg) or the thiol donor N-acetylcysteine (NAC, 150 mg/kg) were administered (p7-p12) or (p7-p17). Vascular endothelial cells were incubated at hyperoxia (40% oxygen) or normoxia (21% oxygen) for 48 h. Vascular density was determined in retinal flat mounts labeled with isolectin B4. Expression of vascular endothelial growth factor, caspase-3, and poly(ADP ribose) polymerase (PARP), activation of Akt and p38 mitogen-activated protein kinase (MAPK), and tyrosine nitration of the phosphatidylinositol (PI) 3-kinase p85 subunit were analyzed by Western blot. Hyperoxia-induced peroxynitrite caused endothelial cell apoptosis as indicated by expression of cleaved caspase-3 and PARP leading to vaso-obliteration. These effects were associated with significant tyrosine nitration of the p85 subunit of PI 3-kinase, decreased Akt activation, and enhanced p38 MAPK activation. Blocking tyrosine nitration of PI 3-kinase with epicatechin or NAC restored Akt phosphorylation, and inhibited vaso-obliteration at p12 and neovascularization at p17 comparable with FeTPPS. Early inhibition of tyrosine nitration with use of epicatechin or NAC can represent safe and effective vascular-protective agents in ischemic retinopathy.

Regeneration Enhances Metastasis: A Novel Role for Neurovascular Signaling in Promoting Melanoma Brain Metastasis.

Neural repair after stroke involves initiation of a cellular proliferative program in the form of angiogenesis, neurogenesis, and molecular growth signals in the surrounding tissue elements. This cellular environment constitutes a niche in which regeneration of new blood vessels and new neurons leads to partial tissue repair after stroke. Cancer metastasis has similar proliferative cellular events in the brain and other organs. Do cancer and CNS tissue repair share similar cellular processes? In this study, we identify a novel role of the regenerative neurovascular niche induced by stroke in promoting brain melanoma metastasis through enhancing cellular interactions with surrounding niche components. Repair-mediated neurovascular signaling induces metastatic cells to express genes crucial to metastasis. Mimicking stroke-like conditions in vitro displays an enhancement of metastatic migration potential and allows for the determination of cell-specific signals produced by the regenerative neurovascular niche. Comparative analysis of both in vitro and in vivo expression profiles reveals a major contribution of endothelial cells in mediating melanoma metastasis. These results point to a previously undiscovered role of the regenerative neurovascular niche in shaping the tumor microenvironment and brain metastatic landscape.

Evaluation of two eco-friendly neutralizers for a spectrum of tissue fixatives for biomedical applications.

Formaldehyde is a widely used aldehyde in biomedical applications, including tissue fixation. It is this same fixative property that can result in toxicity if aldehydes are improperly discarded. A proper neutralization of aldehyde waste products can address this, thereby reducing both health and environmental toxicity concerns. In this study two commercially available products designed to neutralize formaldehyde were evaluated, including neutralization of laboratory derived tissue fixative waste. The primary selection criteria for inclusion in the study were: their ease of use (based on product instructions); the two products assert high levels of formaldehyde neutralization (below 20 ppm) relative to other neutralizing products and their lack of generation of polymeric residues that can clog drains. Both products tested were relatively easy to use and both achieved <10 ppm residual levels of formaldehyde from standard formalin and glutaraldehyde preparations used in research and clinical laboratories.

Cystatin C takes part in melanoma-microglia cross-talk: possible implications for brain metastasis.

The development of melanoma brain metastasis is largely dependent on mutual interactions between the melanoma cells and cells in the brain microenvironment. Here, we report that the extracellular cysteine protease inhibitor cystatin C (CysC) is involved in these interactions. Microglia-derived factors upregulated CysC secretion by melanoma. Similarly, melanoma-derived factors upregulated CysC secretion by microglia. Whereas CysC enhanced melanoma cell migration through a layer of brain endothelial cells, it inhibited the migration of microglia cells toward melanoma cells. CysC was also found to promote the formation of melanoma three-dimensional structures in matrigel. IHC analysis revealed increased expression levels of CysC in the brain of immune-deficient mice bearing xenografted human melanoma brain metastasis compared to the brain of control mice. Based on these in vitro and in vivo experiments we hypothesize that CysC promotes melanoma brain metastasis. Increased expression levels of CysC were detected in the regenerating brain of mice after stroke. Post-stroke brain with melanoma brain metastasis showed an even stronger expression of CysC. The in vitro induction of stroke-like conditions in brain microenvironmental cells increased the levels of CysC in the secretome of microglia cells, but not in the secretome of brain endothelial cells. The similarities between melanoma brain metastasis and stroke with respect to CysC expression by and secretion from microglia cells suggest that CysC may be involved in shared pathways between brain metastasis and post-stroke regeneration. This manifests the tendency of tumor cells to highjack physiological molecular pathways in their progression.

Imaging of Angiotropism/Vascular Co-Option in a Murine Model of Brain Melanoma: Implications for Melanoma Progression along Extravascular Pathways.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

The Akt inhibitor, triciribine, ameliorates chronic hypoxia-induced vascular pruning and TGFß-induced pulmonary fibrosis.

BACKGROUND AND PURPOSE: Interstitial lung disease accounts for a group of chronic and progressive disorders associated with severe pulmonary vascular remodelling, peripheral vascular rarefaction and fibrosis, thus limiting lung function. We have previously shown that Akt is necessary for myofibroblast differentiation, a critical event in organ fibrosis. However, the contributory role of the Akt-mTOR pathway in interstitial lung disease and the therapeutic benefits of targeting Akt and mTOR remain unclear. EXPERIMENTAL APPROACH: We investigated the role of the Akt-mTOR pathway and its downstream molecular mechanisms in chronic hypoxia- and TGFß-induced pulmonary vascular pruning and fibrosis in mice. We also determined the therapeutic benefits of the Akt inhibitor triciribine and the mTOR inhibitor rapamycin for the treatment of pulmonary fibrosis in mice. KEY RESULTS: Akt1(-) (/) (-) mice were protected from chronic hypoxia-induced peripheral vascular pruning. In contrast, hyperactivation of Akt1 induced focal fibrosis similar to TGFß-induced fibrosis. Pharmacological inhibition of Akt, but not the Akt substrate mTOR, inhibited hypoxia- and TGFß-induced pulmonary vascular rarefaction and fibrosis. Mechanistically, we found that Akt1 modulates pulmonary remodelling via regulation of thrombospondin1 (TSP1) expression. Hypoxic Akt1(-) (/) (-) mice lungs expressed less TSP1. Moreover, TSP1(-) (/) (-) mice were resistant to adMyrAkt1-induced pulmonary fibrosis. CONCLUSIONS AND IMPLICATIONS: Our study identified Akt1 as a novel target for the treatment of interstitial lung disease and provides preclinical data on the potential benefits of the Akt inhibitor triciribine for the treatment of interstitial lung disease.

Metformin treatment in the period after stroke prevents nitrative stress and restores angiogenic signaling in the brain in diabetes.

Diabetes impedes vascular repair and causes vasoregression in the brain after stroke, but mechanisms underlying this response are still unclear. We hypothesized that excess peroxynitrite formation in diabetic ischemia/reperfusion (I/R) injury inactivates the p85 subunit of phosphoinositide 3-kinase (PI3K) by nitration and diverts the PI3K-Akt survival signal to the p38-mitogen-activated protein kinase apoptosis pathway. Nitrotyrosine (NY), Akt and p38 activity, p85 nitration, and caspase-3 cleavage were measured in brains from control, diabetic (GK), or metformin-treated GK rats subjected to sham or stroke surgery and in brain microvascular endothelial cells (BMVECs) from Wistar and GK rats subjected to hypoxia/reoxygenation injury. GK rat brains showed increased NY, caspase-3 cleavage, and p38 activation and decreased Akt activation. Metformin attenuated stroke-induced nitrative signaling in GK rats. GK rat BMVECs showed increased basal nitrative stress compared with controls. A second hit by hypoxia/reoxygenation injury dramatically increased the nitration of p85 and activation of p38 but decreased Akt. These effects were associated with impairment of angiogenic response and were restored by treatment with the peroxynitrite scavenger 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride or the nitration inhibitor epicatechin. Our results provide evidence that I/R-induced peroxynitrite inhibits survival, induces apoptosis, and promotes peroxynitrite as a novel therapeutic target for the improvement of reparative angiogenesis after stroke in diabetes.

Angiotropism, pericytic mimicry and extravascular migratory metastasis in melanoma: an alternative to intravascular cancer dissemination.

For more than 15 years, angiotropism in melanoma has been emphasized as a marker of extravascular migration of tumor cells along the abluminal vascular surface, unveiling an alternative mechanism of tumor spread distinct from intravascular dissemination. This mechanism has been termed extravascular migratory metastasis (EVMM). During EVMM, angiotropic tumor cells migrate in a 'pericytic-like' manner (pericytic mimicry) along the external surfaces of vascular channels, without intravasation. Through this pathway, melanoma cells may spread to nearby or more distant sites. Angiotropism is a prognostic factor predicting risk for metastasis in human melanoma, and a marker of EVMM in several experimental models. Importantly, analogies of EVMM and pericytic mimicry include neural crest cell migration, vasculogenesis and angiogenesis, and recent studies have suggested that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes reminiscent of cancer migration and embryonic/stem cell state transitions. A recent work revealed that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression via angiotropism and migration along the abluminal vascular surface. Finally, recent data using imaging of melanoma cells in a murine model have shown the progression of tumor cells along the vascular surfaces. Taken together, these data provide support for the biological phenomenon of angiotropism and EVMM, which may open promising new strategies for reducing or preventing melanoma metastasis.

Cerebral neovascularization and remodeling patterns in two different models of type 2 diabetes.

We previously reported intense pial cerebral collateralization and arteriogenesis in a mild and lean model of type 2 diabetes (T2D), Goto-Kakizaki (GK) rats. Increased cerebral neovascularization differed regionally and was associated with poor vessel wall maturity. Building upon these findings, the goals of this study were to determine whether a) glycemic control prevents this erratic cerebral neovascularization in the GK model, and b) this pathological neovascularization pattern occurs in Lepr(db/db) model, which is the most commonly used model of T2D for studies involving cerebral complications of diabetes. Vascular volume, surface area and structural parameters including microvessel/macrovessel ratio, non-FITC (fluorescein) perfusing vessel abundance, vessel tortuosity, and branch density were measured by 3D reconstruction of FITC stained vasculature in GK rats or Lepr(db/db) mice. GK rats exhibited an increase in all of these parameters, which were prevented by glycemic control with metformin. In Lepr(db/db) mice, microvascular density was increased but there was no change in nonFITC-perfusing vessels. Increased PA branch density was associated with reduced branch diameter. These results suggest that T2D leads to cerebral neovascularization and remodeling but some structural characteristics of newly formed vessels differ between these models of T2D. The prevention of dysfunctional cerebral neovascularization by early glucose control suggests that hyperglycemia is a mediator of this response.

Comparative analysis of the neurovascular injury and functional outcomes in experimental stroke models in diabetic Goto-Kakizaki rats.

Diabetes worsens functional outcome and is associated with greater hemorrhagic transformation (HT) after ischemic stroke. We have shown that diabetic Goto-Kakizaki (GK) rats develop greater HT and neurological deficit despite smaller infarcts after transient middle cerebral artery occlusion (MCAO) with the suture model. However, the impact of (1) the duration of ischemia/reperfusion (I/R); (2) the method of ischemia; and (3) acute glycemic control on neurovascular injury and functional outcome in diabetic stroke remained unanswered. Wistar and GK rats were subjected to variable MCAO by suture or embolus occlusion. A group of GK rats were treated with insulin or metformin before stroke with suture occlusion. In all groups, infarct size, edema, HT occurrence and severity, and functional outcome were measured. Infarct size at 24h was smaller in GK rats with both suture and embolic MCAO, but expanded with longer reperfusion period. Edema and HT were increased in GK rats after 90min and 3h occlusion with the suture model, but not in the embolic MCAO. Neurological deficit was greater in diabetic rats. These findings suggest that diabetes accelerates the development of HT and amplifies vascular damage in the suture model where blood flow is rapidly reestablished. Acute metformin treatment worsened the infarct size, HT, and behavior outcome, whereas insulin treatment showed a protective effect. These results suggest that the impact of ischemia/reperfusion on neurovascular injury and functional outcome especially in disease models needs to be fully characterized using different models of stroke to model the human condition.

Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats.

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.

Enhanced cerebral but not peripheral angiogenesis in the Goto-Kakizaki model of type 2 diabetes involves VEGF and peroxynitrite signaling.

We previously reported enhanced cerebrovascular remodeling and arteriogenesis in experimental type 2 diabetes. This study tested the hypotheses that 1) cerebral but not peripheral angiogenesis is increased in a spatial manner and 2) peroxynitrite orchestrates vascular endothelial growth factor (VEGF)-mediated brain angiogenesis in diabetes. Stereology of brain, eye, and skeletal muscle microvasculature was evaluated in control and diabetic rats using three-dimensional images. Migration and tube formation properties of brain microvascular endothelial cells (BMECs) were analyzed as markers of angiogenesis. Vascular density, volume, and surface area were progressively increased from rostral to caudal sections in both the cerebral cortex and striatum in diabetic rats. Unperfused new vessels were more prominent and the pericyte-to-endothelial cell ratio was decreased in diabetes. Vascularization was greater in the retina but lower in the peripheral circulation. VEGF and nitrotyrosine levels were higher in cerebral microvessels of diabetic animals. Migratory and tube formation properties were enhanced in BMECs from diabetic rats, which also expressed high levels of basal VEGF, nitrotyrosine, and membrane-type (MT1) matrix metalloprotease (MMP). VEGF-neutralizing antibody and inhibitors of peroxynitrite, src kinase, or MMP blocked the migration. Diabetes increases and spatially regulates cerebral neovascularization. Increased VEGF-dependent angiogenic function in BMECs is mediated by peroxynitrite and involves c-src and MT1-MMP activation.

Neurovascular injury in acute hyperglycemia and diabetes: A comparative analysis in experimental stroke.

BACKGROUND: Admission hyperglycemia impacts ischemic stroke deleteriously but the relative role of acute hyperglycemia (HG) versus diabetes in the pathogenesis of this poor outcome is not clear. PURPOSE: To determine the effect of acute HG on neurovascular outcomes of stroke under control and diabetic conditions. METHODS: Moderate acute HG (140-200 mg/dl) was achieved by glucose injection before middle cerebral artery occlusion (MCAO) in control Wistar and diabetic Goto-Kakizaki (GK) rats. Following 3 h MCAO/21 h reperfusion, we measured infarct size, hemorrhagic transformation (HT) frequency, excess hemoglobin, neurobehavioral outcome and plasma and MCA matrix metalloprotease (MMP) activity. RESULTS: Infarct size was significantly smaller in diabetic rats. Moderate acute HG increased neuronal damage in diabetic but not in control rats. HT frequency and hemoglobin were significantly higher in diabetic rats. HG augmented vascular damage in control rats and had no additional effect on bleeding in diabetic rats. Baseline plasma MMP-9 activity was significantly higher in diabetic rats. HG increased MMP-9 activity in control and diabetic rats. Neurological deficit was greater in diabetic rats and was worsened by HG. CONCLUSIONS: The finding that functional outcome is poorer in both acute HG and diabetes without a significant increase in infarct size suggests that amplified vascular damage contributes to neurological deficit in hyperglycemia. These results highlight the importance of vascular protection to improve neurological outcome in acute ischemic stroke.

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.