No abstract

Hyaluronan (HA) is a large nonsulfated glycosaminoglycan and an important regulator of angiogenesis, in particular, the growth and migration of vascular endothelial cells. We have identified some of the key intermediates responsible for induction of mitogenesis and wound recovery. Treatment of bovine aortic endothelial cells with oligosaccharides of hyaluronan (o-HA) resulted in rapid tyrosine phosphorylation and plasma membrane translocation of phospholipase C␥1 (PLC␥1). Cytoplasmic loading with inhibitory antibodies to PLC␥1, G␤, and G␣ i/o/t/z inhibited activation of extracellular-regulated kinase 1/2 (ERK1/2). Treatment with the G␣ i/o inhibitor, pertussis toxin, reduced o-HA-induced PLC␥1 tyrosine phosphorylation, protein kinase C (PKC) ␣ and ␤1/2 membrane translocation, ERK1/2 activation, mitogenesis, and wound recovery, suggesting a mechanism for o-HA-induced angiogenesis through Gproteins, PLC␥1, and PKC. In particular, we demonstrated a possible role for PKC␣ in mitogenesis and PKC␤1/2 in wound recovery. Using antisense oligonucleotides and the Ras farnesylation inhibitor FTI-277, we showed that o-HA-induced bovine aortic endothelial cell proliferation, wound recovery, and ERK1/2 activation were also partially dependent on Ras activation, and that o-HA-stimulated tyrosine phosphorylation of the adapter protein Shc, as well as its association with Sos1. Binding of Src to Shc was required for its activation and for Ras-dependent activation of ERK1/2, cell proliferation, and wound recovery. Neither Src nor Ras activation was inhibited by pertussis toxin, suggesting that their activation was independent of heterotrimeric G-proteins. However, the specific Src kinase inhibitor PP2 inhibited G␤ subunit co-precipitation with PLC␥1, suggesting a possible role for Src in activation of PLC␥1 and interaction between two distinct o-HA-induced signaling pathways.

Background and Purpose —Both vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) are expressed in higher than normal concentrations in the penumbra of patients after ischemic stroke. Because both cytokines are central to the processes of angiogenesis, tissue inflammation, and fibrosis, we performed serial measurements of these cytokines in patients with cerebral infarction and determined their relationship to stroke etiology and volume. Methods —We serially (at days 0, 1, 3, 7, and 14) measured the serum levels of VEGF and active TGF-β1 in 29 patients with acute ischemic stroke. Age-matched healthy subjects (n=26) were used as controls. Results —Expression of VEGF was significantly increased in the majority of patients after acute stroke at each of the time points compared with normal controls. Highest expression occurred at day 7 (588±121 pg/mL; P =0.005), and it remained significantly elevated at 14 days after stroke. Expression of VEGF correlated with infarct volume, clinical disability (Scandinavian Stroke Scale), and peripheral leukocytosis and was significantly higher in patients with atherothrombotic large-vessel disease and ischemic heart disease ( P <0.05 in all cases). In contrast, expression of active TGF-β1 was not significantly different from control patients at any of the measured time points. When the mean concentration of TGF-β1 from each patient (pooled time points) was compared with the control mean, a significant increase was found in only 2 patients, whereas levels decreased in 12 patients ( P <0.05). There was no correlation between circulating active TGF-β1 and VEGF expression, leukocytosis, stroke subtype, or patient disability as assessed by Scandinavian Stroke Scale score. Conclusions —VEGF but not TGF-β1 showed a dramatic increase in serum of stroke patients. Correlation between stroke severity and VEGF concentration suggests it could be involved in the subsequent repair processes resulting in partial recovery after stroke. Correlation between VEGF expression and peripheral leukocytosis suggests that these changes may also reflect the immunologic status of the patient. VEGF may play an important role in the pathophysiology of acute ischemic stroke and could be of value in future treatment strategies.

The extent of recovery from stroke is dependent on the survival of neurons, particularly in peri-infarcted regions. Angiogenesis is critical for the development of new microvessels and leads to re-formation of collateral circulation, reperfusion and better recovery. Hyaluronan (HA) is an important component of the brain extracellular matrix and a regulator of cellular differentiation, migration, proliferation and angiogenesis. We have found that the production of total HA and low molecular mass 3-10 disaccharides of HA (o-HA) was increased in post-mortem tissue and in the serum of patients 1, 3, 7 and 14 days (peaking at 7 days) after ischaemic stroke. Hyaluronidase activity was also increased in serum samples (peaking after 3 days), which might explain the subsequent increase in o-HA. Affinity-histochemical staining was performed using a HA-specific biotinylated binding protein, and it showed enhanced deposition of HA in blood vessels and intracellularly as well as in the nuclei of peri-infarcted neurons. Western blotting and immunohistochemistry demonstrated upregulation of HA synthases (HAS1 and 2) and hyaluronidases (HYAL1 and 2) in inflammatory cells from both stroke and peri-infarcted regions of the brain. HYAL1 was upregulated in microvesssels and intracellularly in neurons, whilst HAS2 became translocated into the nuclei of neurons in peri-infarcted areas. Receptor for HA-mediated motility was observed intracellularly and in the nuclei of neurons, in the tunica media of larger blood vessels and in the endothelial cells of microvessels in stroke-affected tissue, whilst expression of other receptors for HA, CD44 and tumour necrosis factor-stimulated gene 6 (TSG-6) were mainly increased in infiltrating mononuclear cells from inflammatory regions. The data presented here demonstrate that HA breakdown is a feature of the acute stage of stroke injury. Increased o-HA production soon after stroke may be detrimental through enhancement of the inflammatory response, whilst activation of HA and/or o-HA-induced cellular signalling pathways in neurons and microvessels may impact on the remodelling process by stimulating angiogenesis and revascularization, as well as the survival of susceptible neurons.

Recent developments in our understanding of the pathophysiological events that follow acute ischaemic stroke suggest an important role for angiogenesis which, through new blood vessel formation, results in improved collateral circulation and may impact on the medium-to-long term recovery of patients. Future treatment regimens may focus on optimization of this process in the ischaemic boundary zones or 'penumbra' region adjacent to the infarct, where partially affected neurons exposed to intermediate perfusion levels have the capability of survival if perfusion is maintained or normalized. In this review, we present evidence that angiogenesis is a key feature of ischaemic stroke recovery and neuronal post-stroke re-organization, examine the signalling mechanisms through which it occurs, and describe the therapeutic potential of treatments aimed at stimulating revascularization and neuroprotection after stroke.

Hyaluronan (HA), a multifunctional, high molecular weight glycosaminoglycan, is a component of the majority of extracellular matrices. HA is synthesised in a unique manner by a family of hyaluronan synthases, degraded by hyaluronidases and exerts a biological effect by binding to families of cellular receptors, the hyaladhedrins. Receptor binding activates signal pathways in endothelial cells leading to proliferation, migration and differentiation collectively termed angiogenesis. HA and associated enzymes are implicated in the aetiology of cardiovascular disease and cancer and manipulation of HA expression offers a therapeutic target. HA microspheres have been developed as drug delivery agents to deliver HA to sites of disease and also in diagnosis. In this review we discuss some of the recent therapeutic applications of hyaluronan in tissue repair, as a drug delivery system and the synthesis, application and delivery of hyaluronan nanoparticles to target drugs to sites of disease.

Current understanding of the patho-physiological events that follow acute ischaemic stroke suggests that treatment regimens could be improved by manipulation of gene transcription and protein activation, especially in the penumbra region adjacent to the infarct. An immediate reduction in excitotoxicity in response to hypoxia, as well as the subsequent inflammatory response, and beneficial control of reperfusion via collateral revascularization near the ischaemic border, together with greater control over apoptotic cell death, could improve neuronal survival and ultimately patient recovery. Highly significant differences in gene activation between animal models for stroke by middle cerebral artery occlusion, and stroke in patients, may explain why current treatment strategies based on animal models of stroke often fail. We have highlighted the complexities of cellular regulation and demonstrated a requirement for detailed studies examining cell specific protective mechanisms after stroke in humans.

We examined expression of vascular endothelial growth factor (VEGF), phosphorylation of mitogen activated protein kinase (MAP) kinase (ERK1 and ERK2) and tyrosine phosphorylation in 19 patients (aged 58-90 years; mean 75) who died 1-44 days after acute ischaemic stroke. In the grey matter penumbra, 13 of 19 patients showed an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 8-fold, ERK2; 2.2- to 11-fold) compared with normal contralateral tissue. In almost all cases, ERK-2 phosphorylation was higher than ERK1. Of these 13 patients, 11 also showed a general increase in tyrosine kinase phosphorylation, and eight expressed increased levels of VEGF protein (2.5- to 5-fold). In tissue examined directly from the infarct core, activation of the above proteins was not observed in the, majority of patients. In the white matter, seven of 19 patients (penumbra), and nine of 19 patients (stroke) had an increase in MAP kinase tyrosine phosphorylation (ERK1; 2.0- to 4.6-fold and ERK-2; 2.3- to 5.4-fold respectively) compared with normal contralateral tissue. There was no relationship between activation of MAP kinase and expression of VEGF. Examination of phosphorylated MAP kinase by immunohistochemistry revealed an increase in immunoreactivity in neurones, astroglial cells, reactive microglia and endothelial cells in areas surrounding infarcts, especially in areas with the highest density of microvessels. In conclusion, chronic activation of tyrosine phosphorylated events, in particular redistribution and phosphorylation of MAP kinase (ERK1/ERK2) occurs consistently in the grey matter penumbra of brain tissue following ischaemic stroke, and may be associated with increase in expression of VEGF. These signal transduction events could be important determinants of the extent of neuronal survival and/or angiogenic activity in the recovering brain tissue.