The clinical relevance of assessing advanced glycation endproducts accumulation in diabetes.

Cardiovascular disease is the major cause of morbidity and mortality associated with diabetes. There is increasing evidence that advanced glycation endproducts (AGEs) play a pivotal role in atherosclerosis, in particular in diabetes. AGE accumulation is a measure of cumulative metabolic and oxidative stress, and may so represent the "metabolic memory". Furthermore, increased AGE accumulation is closely related to the development of cardiovascular complications in diabetes. This review article will focus on the clinical relevance of measuring AGE accumulation in diabetic patients by focusing on AGE formation, AGEs as predictors of long-term complications, and interventions against AGEs.

Altered vascular responses to circulating angiotensin II in familial combined hyperlipidemia.

OBJECTIVES: Patients with familial combined hyperlipidemia (FCHL) are at increased risk of hypertension and cardiovascular disease. We examined if patients with FCHL have altered microvascular and macrovascular responses to angiotensin II, a principal mediator of the renin-angiotensin-aldosterone system. METHODS: Sixteen patients with FCHL and 16 healthy controls were investigated before, during and after a 3 h intravenous infusion of angiotensin II (10 ng/kg/min). Forearm skin microcirculation was studied by laser Doppler fluxmetry during rest and local heating to 44 degrees C (microvascular hyperemia). RESULTS: Baseline systolic blood pressures were 129 +/- 13 and 123 +/- 12 mmHg in FCHL patients and controls (P = 0.11), respectively. Angiotensin II elicited a greater systolic blood pressure response in the FCHL group (+32 +/- 13 mmHg) than in the control group (+20 +/- 11 mmHg; P < 0.001). At 3 h angiotensin II infusion, microvascular hyperemia increased in the controls (P < 0.001), whereas microvascular hyperemia was unchanged in the FCHL patients (P < 0.01, between groups). CONCLUSION: In healthy individuals, a 3 h intravenous infusion of angiotensin II enhances heat-induced microvascular hyperemia. In FCHL, this microvascular hyperemia is impaired and the systolic blood pressure response is increased. A reduced microvascular dilatation capacity in FCHL may contribute to the observed blood pressure elevation and promote development of micro- and macrovascular complications.

Endothelial function assessment in complicated hypertension.

A large body of evidence indicates that patients with essential hypertension, and even more those with complicated hypertension, are characterized by endothelial dysfunction characterized by impaired NO availability secondary to oxidative stress production. A dysfunctioning endothelium is an early marker of the development of atherosclerotic changes and can also contribute to cardiovascular events. Vascular reactivity tests represent the most widely used methods in the clinical assessment of endothelial function. In the last two decades, many studies have evaluated the endothelium in hypertensive patients, using different techniques. Several methodologies were developed to study microcirculation (resistance arteries and arterioles) and macrocirculation (conduit arteries), both in coronary and peripheral vascular districts. This review will centre on the most relevant available techniques in the research on endothelial dysfunction in essential hypertension, their advantages and limitations, focusing on available data on endothelial dysfunction which characterizes patients with complicated hypertension. No available test to assess endothelial function has sufficient sensitivity and specificity to be used in clinical practice. Therefore, the optimal methodology for investigating the multifaceted aspects of endothelial dysfunction is still under debate. Only the growing concordant results from different reproducible and reliable methods exploring endothelial function with different stimuli will support and strengthen experimental findings, thus providing conclusive answers in this area of research.

Diabetes, cognitive function, and the blood-brain barrier.

From a complications standpoint, diabetes mellitus is a disease of the vasculature. Diabetics face a considerably higher risk of developing cardiovascular and cerebrovascular diseases. Both large and small blood vessels are susceptible to alterations from diabetes. Endothelial cell dysfunction associated with small vessel (known as microangiopathy) is a primary factor in the development and progression of diabetes-related disabilities, including blindness, kidney failure, and peripheral neuropathy. Recent clinical evidence show that people with diabetes have increased incidences of vascular dementia, ventricular hypertrophy, lacunar infarcts, hemorrhage, and may be a predisposing factor for Alzheimer's disease. However, the effects of diabetes mellitus on the cerebral microvascular are still largely unknown. This communication will review the relationship between diabetes mellitus and changes in cognition with a particular focus on how alterations in blood-brain barrier structure and function may play a long term role in worsened cognitive abilities.

Differential expression of the multidrug resistance-related proteins ABCb1 and ABCc1 between blood-brain interfaces.

Cerebral homeostasis results from the presence of the protective blood-brain and blood-cerebrospinal fluid barriers located respectively at the brain capillary endothelium and the choroid plexus epithelium. ABCb1 (Pgp) and ABCc1 (Mrp1) transporters are two major proteins of neuroprotection whose localization and functional significance at both barriers remain partly unsettled. We conducted a comparative analysis of their relative protein content between the two blood-brain interfaces. Microvessels and choroid plexuses located in the fourth and lateral ventricles were isolated from developing and adult rat brains, and whole homogenates were submitted to quantitative Western blot analysis by using standard curves generated from one of the samples. In adult, choroid plexus-associated Pgp content was less than 0.5% of the level in microvessels, whereas Mrp1 content in microvessels was 4% of that in the fourth ventricle choroid plexus. Pgp but not Mrp1 was enriched in microvessels over parenchyma. In choroid plexuses, Mrp1 displayed a basolateral epithelial localization, and reached its high adult protein level, early during postnatal development. In postnatal as in adult microvessels, Pgp localization appeared luminal. However, by contrast to Mrp1, the level of this transporter increased 4.6-fold between 9-day-old and adult animals. Western blot analysis of human samples confirmed the mirror image of Pgp and Mrp1 expression between the two barriers. We conclude that there are major differences in the mechanisms by which blood-brain interfaces fulfill their neuroprotective functions. The data also highlight the significance of the neuroprotective function of the choroid plexus during brain maturation, when the microvasculature is still developing.

Protein components of the blood-brain barrier (BBB) in the mediobasal hypothalamus.

The blood-brain barrier (BBB) plays an important role in controlling the access of substances to the brain. Of the circumventricular organs (CVO), i.e. areas that lack a BBB, the median eminence and its close relationship with the hypothalamic arcuate nucleus plays an important role in controlling the entry of blood-borne substances to neurons of the mediobasal hypothalamus. In order to clarify the nature of the BBB in the median eminence-arcuate nucleus complex, we have used immunohistochemistry and antisera to protein components of the BBB-(1) tight junctions, claudin-5 and zona occludens-1 (ZO-1); (2) endothelial cells: (a) all endothelial cells: rat endothelial cell antigen-1 (RECA-1), (b) endothelial cells at BBB: endothelial barrier antigen (EBA), glucose transporter 1 (GLUT1) and transferrin receptor (TfR), and (c) endothelial cells at CVOs: dysferlin; (3) basal lamina: laminin; (4) vascular smooth muscle cells: smooth muscle actin (SMA); (5) pericytes: chondroitin sulfate proteoglycan (NG2); (6) glial cells: (a) astrocytes: glial fibrillary acidic protein (GFAP), (b) tanycytes: dopamine- and cAMP-regulated phosphoprotein of 32kDA (DARPP-32), (c) microglia: CD11b. Neuronal cell bodies located in the ventromedial aspect of the arcuate nucleus were visualized by antiserum to agouti-related protein (AgRP). The study provides a detailed analysis on the cellular localization of BBB components in the mediobasal hypothalamus. Some vessels in the ventromedial aspect of the arcuate nucleus lacked the BBB markers EBA and TfR, suggesting an absence of an intact BBB. These vessels may represent a route of entry for circulating substances to a subpopulation of arcuate nucleus neurons.

Occludin oligomeric assembly at tight junctions of the blood-brain barrier is disrupted by peripheral inflammatory hyperalgesia.

Tight junctions (TJs) at the blood-brain barrier (BBB) dynamically alter paracellular diffusion of blood-borne substances from the peripheral circulation to the CNS in response to external stressors, such as pain, inflammation, and hypoxia. In this study, we investigated the effect of lambda-carrageenan-induced peripheral inflammatory pain (i.e., hyperalgesia) on the oligomeric assembly of the key TJ transmembrane protein, occludin. Oligomerization of integral membrane proteins is a critical step in TJ complex assembly that enables the generation of tightly packed, large multiprotein complexes capable of physically obliterating the interendothelial space to inhibit paracellular diffusion. Intact microvessels isolated from rat brains were fractionated by detergent-free density gradient centrifugation, and gradient fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/ Western blot. Injection of lambda-carrageenan into the rat hind paw produced after 3 h a marked change in the relative amounts of oligomeric, dimeric, and monomeric occludin isoforms associated with different plasma membrane lipid raft domains and intracellular compartments in endothelial cells at the BBB. Our findings suggest that increased BBB permeability (i.e., leak) associated with lambda-carrageenan-induced peripheral inflammatory pain is promoted by the disruption of disulfide-bonded occludin oligomeric assemblies, which renders them incapable of forming an impermeant physical barrier to paracellular transport.

Shear stress-induced Ets-1 modulates protease inhibitor expression in microvascular endothelial cells.

Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4, and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm(2)) for 0.5, 2, and 24 h. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2 h shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1, and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.

Pathophysiological role of platelets and platelet system in acute pancreatitis.

The most successful approach for restoring normal long-term glucose homeostasis in type I diabetes mellitus is whole-organ pancreas transplantation. Graft pancreatitis is observed in up to 20% of patients and may lead to loss of the transplanted organ. Several pathophysiological events have been implicated in this form of pancreatitis. The most important cause of early graft pancreatitis is ischemia/reperfusion (I/R)-related disturbance of microvascular perfusion with subsequent hypoxic tissue damage. Recently, considerable evidence accumulated that, among a variety of other pathophysiological events, the activation of platelets can contribute to I/R injury in the course of acute pancreatitis experimentally and clinically. This review summarizes the events affecting platelet function and, therefore, pancreatic microcirculation leading to acute pancreatitis. Therapeutic approaches and own results are presented.

Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses.

Mechanical stretch, an important growth stimulus, results not only from pulsatile blood flow and diastolic stretch of the ventricles [cyclic stretch (CS)] but also from tissue expansion during growth [constant static stretch (SS)]. We compared growth factor receptor expression and vasculogenic/angiogenic responses of rat coronary microvascular endothelial cells (ECs) by exposing cells to CS (10% elongation at 30 cycles/min) and SS (constant 10% elongation). Both CS and SS increased VEGF receptor (VEGF-R)2 protein levels and the extent of tube formation and branching. Moreover, both CS and SS enhanced VEGF-induced cell proliferation and tube formation, indicating that both types of stretch increase the sensitivity of ECs to VEGF. Blockade of VEGF-R2 prevented the increases in EC proliferation and aggregate tube length. However, CS but not SS enhanced EC Tie-2 protein and migration. CS affected a greater increase in tube length and branch formation than did SS. A unique finding was that SS but not CS increased VEGFR-1 in ECs. Our study is the first to distinguish between the effects of CS and SS on growth factor receptor expression and rat coronary microvascular EC proliferation, migration, and tube formation. In conclusion, EC angiogenic responses to these two types of stretch display both differences and similarities, but both CS and SS are dependent on VEGF-R2 signaling for their vasculogenic/angiogenic effects.

Lipopolysaccharide plus hypoxia and reoxygenation synergistically reduce electrical coupling between microvascular endothelial cells by dephosphorylating connexin40.

We showed that lipopolysaccharide (LPS) or hypoxia and reoxygenation (H/R) decreases electrical coupling between microvascular endothelial cells by targeting the gap junction protein connexin40 (Cx40), tyrosine kinase-, ERK1/2-, and PKA-dependently. Since LPS can compromise microvascular blood flow, resulting in micro-regional H/R, the concurrent LPS + H/R could reduce coupling to a much greater extent than LPS or H/R alone. We examined this possibility in a model of cultured microvascular endothelial cells (mouse skeletal muscle origin) in terms of electrical coupling and the phosphorylation status of Cx40. To assess coupling, we measured the spread of electrical current injected into the cell monolayer and computed the intercellular resistance as an inversed measure of coupling. In wild type cells, but not in Cx40 null cells, concurrent LPS + H/R synergistically increased resistance by approximately 270%, well above the level observed for LPS or H/R alone. Cx37 and Cx43 protein expression did not differ between Cx40 null and wild type cells. LPS + H/R increased resistance PKA- and PKC-dependently. By immunoprecipitating Cx40, we found that LPS + H/R reduced serine phosphorylation to a much greater degree than that observed for LPS or H/R alone. Further, PKA-specific, but not PKC-specific serine phosphorylation of Cx40 was also significantly reduced following LPS + H/R. This reduction was prevented by tyrosine kinase and MEK1/2 inhibition, by PKA activation, and mimicked in control cells by PKA inhibition. We conclude that LPS + H/R initiates tyrosine kinase- and ERK1/2-sensitive signaling that synergistically reduces inter-endothelial electrical coupling by dephosphorylating PKA-specific serine residues of Cx40.

Antigen presentation by human microvascular endothelial cells triggers ICAM-1-dependent transendothelial protrusion by, and fractalkine-dependent transendothelial migration of, effector memory CD4+ T cells.

TCR engagement on adherent human effector memory CD4(+) T cells by TNF-treated HUVECs under flow induces formation of a transendothelial protrusion (TEP) by the T cell but fails to induce transendothelial migration (TEM). In contrast, TCR engagement of the same T cell populations by TNF-treated human dermal microvascular cells (HDMEC) not only induces TEP formation, but triggers TEM at or near the interendothelial cell junctions via a process in which TEP formation appears to be the first step. Transduction of adhesion molecules in unactivated HDMEC and use of blocking Abs as conducted with TNF-activated HDMEC indicate that ICAM-1 plays a nonredundant role in TCR-driven TEP formation and TEM, and that TCR-driven TEM is also dependent upon fractalkine. TEP formation, dependence on ICAM-1, and dependence on fractalkine distinguish TCR-induced TEM from IP-10-induced TEM. These in vitro observations suggest that presentation of Ag by human microvascular endothelial cells to circulating CD4(+) effector memory T cells may function to initiate recall responses in peripheral tissues.

Disruption of Ang-1/Tie-2 signaling contributes to the impaired myocardial vascular maturation and angiogenesis in type II diabetic mice.

OBJECTIVE: Microvascular insufficiency represents a major cause of end-organ failure among diabetics. The current studies were undertaken to determine whether dysregulation of the angiopoietins/Tie-2 system would result in an impairment of smooth muscle cell (SMC) recruitment and vascular maturation, which contributes to impaired angiogenesis in diabetes. METHODS AND RESULTS: Tie-2 expression was significantly attenuated, whereas angiopoietin-2 (Ang-2) was increased in db/db mice subjected to myocardial ischemia. Our morphological analysis showed that the number of SMC coverage area per neovessel was significantly reduced in db/db mice. This was accompanied by a significant reduction of myocardial capillary density and arteriole formation. Interestingly, Angiopoietin-1(Ang-1)-induced SMC recruitment and vessel outgrowth were severely impaired in db/db mice. Our in vitro studies further demonstrated that exposure of mouse heart endothelial cells to high glucose resulted in a significant upregulation of Ang-2 and a downregulation of Tie-2 expression. These alterations led to a significant impairment of Ang-1-induced Akt and eNOS phosphorylation, along with a remarkable impairment of Ang-1-induced endothelial cell migration and endothelial cell spheroid sprouting. Ang-1 gene transfer restored Tie-2 expression and rescued these abnormalities in diabetes. CONCLUSIONS: Our findings underscore the important role of Ang-1-Tie-2 signaling in the diabetes-induced impairment of vascular maturation and angiogenesis.

Carbon monoxide and nitric oxide mediate cytoskeletal reorganization in microvascular cells via vasodilator-stimulated phosphoprotein phosphorylation: evidence for blunted responsiveness in diabetes.

OBJECTIVE: We examined the effect of the vasoactive agents carbon monoxide (CO) and nitric oxide (NO) : n the phosphorylation and intracellular redistribution of vasodilator-stimulated phosphoprotein (VASP), a critical actin motor protein required for cell migration that also controls vasodilation and platelet aggregation. RESEARCH DESIGN AND METHODS: We examined the effect of donor-released CO and NO in endothelial progenitor cells (EPCs) and platelets from nondiabetic and diabetic subjects and in human microvascular endothelial cells (HMECs) cultured under low (5.5 mmol/l) or high (25 mmol/l) glucose conditions. VASP phosphorylation was evaluated using phosphorylation site-specific antibodies. RESULTS: In control platelets, CO selectively promotes phosphorylation at VASP Ser-157, whereas NO promotes phosphorylation primarily at Ser-157 and also at Ser-239, with maximal responses at 1 min with both agents on Ser-157 and at 15 min on Ser-239 with NO treatment. In diabetic platelets, neither agent resulted in VASP phosphorylation. In nondiabetic EPCs, NO and CO increased phosphorylation at Ser-239 and Ser-157, respectively, but this response was markedly reduced in diabetic EPCs. In endothelial cells cultured under low glucose conditions, both CO and NO induced phosphorylation at Ser-157 and Ser-239; however, this response was completely lost when cells were cultured under high glucose conditions. In control EPCs and in HMECs exposed to low glucose, VASP was redistributed to filopodia-like structures following CO or NO exposure; however, redistribution was dramatically attenuated under high glucose conditions. CONCLUSIONS: Vasoactive gases CO and NO promote cytoskeletal changes through site- and cell type-specific VASP phosphorylation, and in diabetes, blunted responses to these agents may lead to reduced vascular repair and tissue perfusion.

Modulation of P-glycoprotein at the blood-brain barrier: opportunities to improve central nervous system pharmacotherapy.

Pharmacotherapy of central nervous system (CNS) disorders (e.g., neurodegenerative diseases, epilepsy, brain cancer, and neuro-AIDS) is limited by the blood-brain barrier. P-glycoprotein, an ATP-driven, drug efflux transporter, is a critical element of that barrier. High level of expression, luminal membrane location, multispecificity, and high transport potency make P-glycoprotein a selective gatekeeper of the blood-brain barrier and thus a primary obstacle to drug delivery into the brain. As such, P-glycoprotein limits entry into the CNS for a large number of prescribed drugs, contributes to the poor success rate of CNS drug candidates, and probably contributes to patient-to-patient variability in response to CNS pharmacotherapy. Modulating P-glycoprotein could therefore improve drug delivery into the brain. Here we review the current understanding of signaling mechanisms responsible for the modulation of P-glycoprotein activity/expression at the blood-brain barrier with an emphasis on recent studies from our laboratories. Using intact brain capillaries from rats and mice, we have identified multiple extracellular and intracellular signals that regulate this transporter; several signaling pathways have been mapped. Three pathways are triggered by elements of the brain's innate immune response, one by glutamate, one by xenobiotic-nuclear receptor (pregnane X receptor) interactions, and one by elevated beta-amyloid levels. Signaling is complex, with several pathways sharing common signaling elements [tumor necrosis factor (TNF) receptor 1, endothelin (ET) B receptor, protein kinase C, and nitric-oxide synthase), suggesting a regulatory network. Several pathways include autocrine/paracrine elements, involving release of the proinflammatory cytokine, TNF-alpha, and the polypeptide hormone, ET-1. Finally, several steps in signaling are potential therapeutic targets that could be used to modulate P-glycoprotein activity in the clinic.

Monitoring oxidative stress in vascular endothelial cells in response to fluid shear stress: from biochemical analyses to micro- and nanotechnologies.

Hemodynamics, specifically, fluid shear stress, modulates the focal nature of atherosclerosis. Shear stress induces vascular oxidative stress via the activation of membrane-bound NADPH oxidases present in vascular smooth muscle cells, fibroblasts, and phagocytic mononuclear cells. Shear stress acting on the endothelial cells at arterial bifurcations or branching points regulates both NADPH oxidase and nitric oxide (NO) synthase activities. The former is considered a major source of oxygen-centered radicals (i.e., superoxide anion [O2(.-)]) that give rise to oxidative stress; the latter is a source of nitrogen-centered radicals (i.e., nitric oxide [NO]) that give rise to nitrative/nitrosative stress. In addition to conventional biochemical analyses, the emerging microelectromechanical systems (MEMS) provide spatial and temporal resolutions to investigate the mechanisms whereby the characteristics of shear stress regulate the biological activities of endothelial cells at the complicated arterial geometry. In parallel, the development of MEMS liquid chromatography (LC) provides a new venue to measure circulating oxidized low-density lipoprotein (ox-LDL) particles as a lab-on-a chip platform. Nanowire-based field effect transistors further pave the way for a high throughput approach to analyze the LDL redox state. Integration of MEMS with oxidative biology is synergistic in assessing vascular oxidative stress. The MEMS LC provides an emerging lab-on-a-chip platform for ox-LDL analysis. In this context, this chapter has integrated expertise from the fields of vascular biology and oxidative biology to address the dynamics of inflammatory responses.

Differential effects of GdCl3- or MDP treatment on rat liver microcirculation and gene expression in the hepatic non-parenchymal cell fraction in LPS shock.

BACKGROUND: Dichloromethylenebisphosphonate (MDP) and gadolinium chloride (GdCl(3)) are substances frequently used for experimental depletion of Kupffer Cells (KC) in models of endotoxin shock. The aim was to determine whether depletion of KC through pretreatment with GdCl(3) or MDP alters the hepatic microcirculation during lipopolysaccharide (LPS)-induced shock in rats and to test if there are substance-specific differences. METHODS: Rats received either MDP or GdCl(3) or saline prior to induction of LPS shock. Hepatic microcirculation was evaluated by intravital microscopy (sinusoidal diameter, sinusoidal bloodflow, leukocyte adhesion), and the gene expression in the hepatic non-parenchymal cell fraction was determined by RT-PCR. RESULTS: GdCl(3) pretreatment prevented sinusoidal narrowing but did not restore sinusoidal blood flow and did not normalize leukocyte-endothelial interaction time after LPS. In contrast, MDP pretreatment improved hepatic microcirculation consistently for all parameters measured compared to GdCl(3) pretreated animals. In the non-parenchymal cell fraction, eNOS gene expression was preserved and gene expression of TNF-alpha was blocked after MDP but not after GdCl(3) application prior to LPS shock. CONCLUSIONS: The results show that GdCl(3) and MDP cannot be used equivalently for experimental KC depletion in the condition of LPS-induced shock. These findings should be taken into consideration in studies that evaluate the role of Kupffer cells in models of endotoxin-induced shock.

Resveratrol attenuates ischemic brain damage in the delayed phase after stroke and induces messenger RNA and protein express for angiogenic factors.

BACKGROUND: It has been reported recently that resveratrol preconditioning can protect the brain from ischemia-reperfusion injury. However, it was unclear whether resveratrol administration after stroke was beneficial to the delayed phases after focal cerebral ischemia injury. This study investigated the effects and possible protective mechanism of resveratrol on the delayed phase after focal cerebral ischemia injury in mice. METHODS: Mice were randomly assigned to five groups according to the time of administration of resveratrol. Control group mice received a corresponding volume of saline solution (0.9% NaCl) containing 20% hydroxypropyl h-cyclodextrin by gavage and were exposed to middle cerebral artery (MCA) occlusion and reperfusion injury. The treatment groups received resveratrol (50 mg/kg/d, gavage) until day 7. Ischemia group mice received their first dose 5 minutes before MCA ischemia, reperfusion group mice received their first dose 5 minutes before MCA reperfusion, first-day, group mice received their first dose 24 hours after MCA reperfusion, and third-day group mice received their first dose at 72 hours after MCA reperfusion. Brain injury was evaluated by triphenyltetrazolium chloride staining and neurologic examination 7 days after reperfusion. The microvascular cell number was examined with immunohistochemistry staining. Effect of resveratrol on matrix metalloproteinase-2 (MMP-2) and vascular endothelial growth factor (VEGF) gene expression was investigated with reverse transcriptase-polymerase chain reaction and Western blot. RESULTS: The mean neurologic scores and infarct volumes of the ischemia and reperfusion groups were lower than that of the control group at 7 days after MCA reperfusion (P < .05). Immunohistochemistry staining showed significantly less reduction in the number of microvessels in the cortical area of mice of the ischemia and reperfusion groups compared with controls. The ischemic hemispheres of the ischemia and reperfusion groups showed significantly (P < .05) elevated levels of protein of MMP-2 and VEGF. CONCLUSIONS: Resveratrol administration by gavage provided an important neuroprotective effect on focal cerebral ischemic injury in the delayed phase. The elevated MMP-2 and VEGF levels might be important in the neuroprotective effect of resveratrol administration by inducing angiogenesis.