Feasibility and Safety of Intra-arterial Pericyte Progenitor Cell Delivery Following Mannitol-Induced Transient Blood-Brain Barrier Opening in a Canine Model.

Stem cell therapy is currently being studied with a view to rescuing various neurological diseases. Such studies require not only the discovery of potent candidate cells but also the development of methods that allow optimal delivery of those candidates to the brain tissues. Given that the blood-brain barrier (BBB) precludes cells from entering the brain, the present study was designed to test whether hyperosmolar mannitol securely opens the BBB and enhances intra-arterial cell delivery. A noninjured normal canine model in which the BBB was presumed to be closed was used to evaluate the feasibility and safety of the tested protocol. Autologous adipose tissue-derived pericytes with platelet-derived growth factor receptor ß positivity were utilized. Cells were administered 5 min after mannitol pretreatment using one of following techniques: (1) bolus injection of a concentrated suspension, (2) continuous infusion of a diluted suspension, or (3) bolus injection of a concentrated suspension that had been shaken by repeated syringe pumping. Animals administered a concentrated cell suspension without mannitol pretreatment served as a control group. Vital signs, blood parameters, neurologic status, and major artery patency were kept stable throughout the experiment and the 1-month posttreatment period. Although ischemic lesions were noted on magnetic resonance imaging in several mongrel dogs with concentrated cell suspension, the injection technique using repeated syringe shaking could avert this complication. The cells were detected in both ipsilateral and contralateral cortices and were more frequent at the ipsilateral and frontal locations, whereas very few cells were observed anywhere in the brain when mannitol was not preinjected. These data suggest that intra-arterial cell infusion with mannitol pretreatment is a feasible and safe therapeutic approach in stable brain diseases such as chronic stroke.

Suitability of autologous serum for expanding rabbit adipose-derived stem cell populations.

Adipose-derived stem cells (ASCs) are believed to have potential use for treating many illnesses. Most cells, including ASCs, are generally cultured in medium containing fetal bovine serum (FBS). However, FBS, which could induce an immune response or infection, is not recommended for clinical applications. In the present study, we evaluated the morphology, proliferation rate, and characterization of rabbit ASCs grown in medium containing autologous serum (AS) and compared these cells to ones cultured with FBS. Morphological changes were monitored by microscopy and flow cytometry. Proliferation rates were assessed with cell counting and ASC phenotypes were characterized by flow cytometry using representative surface markers (CD44 and CD45). Expression of epidermal growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor was measured by reverse transcription-polymerase chain reaction. Results of our study showed that ASCs had a greater expansion rate in AS without developing morphological heterogeneity than cells grown in FBS. AS-cultured ASCs expressed representative growth factors, CD44 but not CD45, similar to cells cultured in FBS. Expression levels of some growth factors were different between AS and FBS. In conclusion, our findings indicated that AS could potentially be used as a culture medium supplement for the expansion of autologous ASCs.

Circulating CD62E+ microparticles and cardiovascular outcomes.

BACKGROUND: Activated endothelial cells release plasma membrane submicron vesicles expressing CD62E (E-selectin) into blood, known as endothelial microparticles (EMPs). We studied whether the levels of endothelial microparticles expressing CD62E(+), CD31(+)/Annexin-V(+), or CD31(+)/CD42(-) predict cardiovascular outcomes in patients with stroke history. METHODS/PRINCIPAL FINDINGS: Patients with stroke history at least 3 months prior to enrolment were recruited. Peripheral blood EMP levels were measured by flow cytometry. Major cardiovascular events and death were monitored for 36 months. Three hundred patients were enrolled, of which 298 completed the study according to protocol. Major cardiovascular events occurred in 29 patients (9.7%). Nine patients died, five from cardiovascular causes. Cumulative event-free survival rates were lower in patients with high levels of CD62E(+) microparticles. Multivariate Cox regression analysis adjusted for cardiovascular risk factors, medications and stroke etiologic groups showed an association between a high CD62E(+) microparticle level and a risk of major cardiovascular events and hospitalization. Levels of other kinds of EMPs expressing CD31(+)/Annexin-V(+) or CD31(+)/CD42(-) markers were not predictive of cardiovascular outcomes. CONCLUSION: A high level of CD62E(+) microparticles is associated with cardiovascular events in patients with stroke history, suggesting that the systemic endothelial activation increases the risk for cardiovascular morbidities.

Risk of macrovascular complications in type 2 diabetes mellitus: endothelial microparticle profiles.

BACKGROUND: While the adverse impact of diabetes on microvessels is well known, the risks of macroangiopathy are less well recognized. Here, we determine the differential risk and endothelial microparticle (EMP) profile of vascular complications in type 2 diabetes mellitus. METHODS: Macroangiopathy was evaluated for cerebrovascular disease, coronary artery disease and peripheral artery disease; microangiopathy was evaluated for retinopathy, nephropathy and peripheral neuropathy. Clinical and laboratory factors were compared among 149 patients with no vascular complications or with microangiopathic and/or macroangiopathic complications. EMPs were also examined by flow cytometry using CD31, CD42b, annexin V (AV), and CD62E antibodies in the peripheral blood of patients. RESULTS: Diabetes of long duration, an elevated hemoglobin A1c (HbA1c) and concomitant hypertension were significantly associated with the occurrence of vascular complications. Dyslipidemia and a high body mass index were significantly associated with macroangiopathy, while diabetes of long duration and a high concentration of HbA1c were associated with microangiopathy. The EMP (CD31+/CD42b-, CD31+/AV+) levels were higher in patients with macroangiopathy than in patients with microangiopathy and no complications. The EMP level was also independently associated with macroangiopathy in diabetic patients. CONCLUSIONS: Microangiopathy and macroangiopathy in diabetic patients appear to have a different pathophysiological basis. The measurement of EMP would be helpful to differentiate the risk of diabetic vascular complications.

Multipotent PDGFRß-expressing cells in the circulation of stroke patients.

Tissue pericytes respond to injury, and support vascular and tissue regeneration. The presence of pericytes in the circulation may provide an attractive framework for tissue regeneration. Here, we detected multipotent pericyte-like cells in the circulating blood and determined its profiles during cerebral ischemia. Pericyte-like cells were isolated from the peripheral blood of acute stroke patients or asymptomatic individuals with vascular risk factors by fluorescence or magnetic activated cell sorting with anti-PDGF receptor-beta (PDGFRß) antibody. The morphologic and molecular features of circulating PDGFRß(+) cells were compared with tissue pericytes, and the associations with respect to quantity in the blood, culture outcome, and patient characteristics were analyzed. We found an increase in circulating PDGFRß(+) cells in acute stroke patients compared to controls and a correlation with neurologic impairment. The isolated PDGFRß(+) cells expressed mesenchymal stem cell markers, proliferated, and were multipotent under permissive culture conditions. The multipotent nature of these cells was comparable to fat-derived PDGFRß(+) cells. These cells could be obtained by pharmacologic stimulation using bone marrow mobilizer. Circulating PDGFRß(+) cells will be useful for future research involving endogenous recovery or autologous cell-based therapy.

Dysfunctional characteristics of circulating angiogenic cells in Alzheimer's disease.

Vascular senescence contributes to the progression of Alzheimer's disease (AD) and circulating angiogenic cells (CACs) participate in the maintenance of the endothelium. As a step toward the development endothelial regeneration therapies for AD, we investigated the functional characteristics of CACs in AD patients. We enrolled AD patients and non-demented risk factor control subjects after matching for age, sex, and Framingham risk score. CACs were cultured from peripheral blood samples taken from subjects and used for various ex vivo assays. CACs from AD patients showed reduced chemotaxis, increased senescence, reduced paracrine angiogenic activity, and altered gene expression patterns compared to CACs from risk factor (RF) controls. Addition of high concentration Abeta{1-42} (200, 2000 ng/mL) to the CAC culture reduced CAC counts and endothelial nitric oxide synthase/Akt phosphorylation and induced apoptosis. However, lower concentration of Abeta{1-42} (2, 20 ng/mL) failed to reduce the CAC counts. CACs from AD patients were more susceptible to the cytotoxic effect of Abeta{1-42} than CACs from RF controls. In summary, AD patients have intrinsic dysfunctions of CACs which provides an extended understanding of vascular endothelial pathogenesis in AD.

Circulating endothelial microparticles as a marker of cerebrovascular disease.

OBJECTIVE: Circulating endothelial microparticles (EMPs) have been reported to reflect vascular damage. Detailed profiling of these blood endothelial markers may adumbrate the pathogenesis of stroke or enable determination of the risk for stroke. We investigated EMP profiles in patients at risk for cerebrovascular disease. METHODS: We prospectively examined 348 consecutive patients: 73 patients with acute stroke and 275 patients with vascular risk factors but no stroke events. We quantified various types of EMPs by flow cytometry using CD31, CD42b, annexin V (AV), and CD62E antibodies in the peripheral blood of patients. This method allowed fractionation of CD31(+)/CD42b(-), CD31(+)/AV(+), and CD62E(+) EMPs. Clinical and laboratory factors associated with EMPs were assessed. RESULTS: Recent ischemic episodes were found to be more strongly associated with greater CD62E(+) EMP levels than with levels of other phenotypes. Increased National Institutes of Health Stroke Scale scores and infarct volumes in acute stroke patients were significantly associated with greater CD62E(+) EMP levels. In the risk factor group, patients with extracranial arterial stenosis had greater CD62E(+) EMP levels, whereas those with intracranial arterial stenosis had greater CD31(+)/CD42b(-) and CD31(+)/AV(+) EMP levels. The ratio of CD62E(+) to CD31(+)/CD42b(-) or CD31(+)/AV(+) EMP level significantly discriminated extracranial and intracranial arterial stenosis. INTERPRETATION: Circulating EMP phenotypic profiles reflect distinct phenotypes of cerebrovascular disease and are markers of vascular pathology and an increased risk for ischemic stroke.

Circulating endothelial progenitor cells as a pathogenetic marker of moyamoya disease.

Moyamoya disease (MMD) is an unusual form of chronic cerebrovascular occlusive disease that involves the formation of characteristically abnormal vessels. Recent studies have reported that colony-forming unit (CFU) and outgrowth cells represent a subpopulation of endothelial progenitor cells (EPCs). Here, we attempted to determine the significance of CFU number and outgrowth cell yield in MMD. Endothelial progenitor cells were isolated from the blood of 24 adult MMD patients and from 48 age- and risk factor-matched control subjects. After 7 days of culture, CFUs were determined, and yields of outgrowth cells were measured during 2 months of culture. The EPC function was also evaluated using matrigel plate assays. It was found that CFU numbers were significantly lower in MMD patients than in controls. Moreover, during long-term culture, outgrowth cells were isolated from only 10% of control subjects but from 33% of MMD patients, and CFU numbers and tube formation were found to be lower in advanced MMD cases than in those with early stage disease, whereas outgrowth cells were more frequently detected in those with early MMD and moyamoya vessels than in those with advanced disease. These characteristics of circulating EPCs reflect mixed conditions of vascular occlusion and abnormal vasculogenesis during the pathogenesis of MMD.

Hypoxic activation of unoccupied estrogen-receptor-alpha is mediated by hypoxia-inducible factor-1 alpha.

The estrogen receptor (ER) plays an important role in breast cancer development and progression. Hypoxia has been shown to modulate the level of ERalpha expression, which is intimately associated with the biology of breast carcinomas. However, the effect of hypoxia on ERalpha-mediated transactivation is largely unknown. In this report, we have examined ligand-independent transcriptional activation of ERalpha by hypoxia. The hypoxia-induced ERalpha-mediated transcriptional response was inhibited by the ER antagonist ICI 182,780 as determined by transient expression of ERalpha and ER-responsive reporter plasmids in the HEK 293 cells. Hypoxic activation of ERalpha was dependent on the increased expression of hypoxia-inducible factor-1alpha (HIF-1alpha), as examined in HEK 293 cells under conditions of normoxia. These results indicate that hypoxia activates ERalpha in a ligand-independent manner, possibly through the interaction between HIF-1alpha and ERalpha.

Hypertonic sodium chloride induction of cyclooxygenase-2 occurs independently of NF-kappaB and is inhibited by the glucocorticoid receptor in A549 cells.

Cellular response to a hypertonic environment is important for fluid clearance in the lung. Hypertonicity modulates prostaglandin synthesis by influencing cyclooxygenase-2 (COX-2) expression in tissues such as liver and kidney via a mitogen-activated protein kinase (MAPK)-dependent pathway. However, little is known about COX-2 expression in response to hypertonicity in the lung. COX-2 mRNA accumulation induced by hypertonic NaCl was detected after 1 h of treatment, and COX-2 mRNA continued to accumulate until 18 h, the longest time point examined, in human alveolar epithelial A549 cells. This induction was a transcriptional event that occurred in the absence of the protein synthesis inhibitor cycloheximide and was the result of enhanced promoter activity, as examined with the use of full-length COX-2 promoter-driven reporter plasmids. The induction of COX-2 expression by hypertonic NaCl did not require the activation of NF-kappaB. The p38 MAPK inhibitor, SB203580, or MEK1/2 inhibitor, U0126, inhibited hypertonic induction of COX-2 expression. We examined whether the hypertonic induction of COX-2 was under the influence of glucocorticoid; we found that COX-2 promoter activity and mRNA and protein levels were depressed by dexamethasone and antagonized by the glucocorticoid receptor (GR) antagonist RU486. Our data demonstrate that the induction of COX-2 expression by hypertonic NaCl occurs independently of NF-kappaB and is inhibited by the GR in A549 cells.