Santhanam, Anantha Vijay R.

Evidence continues to accumulate on the importance of endothelial progenitor cells (EPCs) in neovascularization of ischemic tissues and repair of injured endothelium. The present study was designed to determine the in vivo effect of EPCs on the vasomotor function of rabbit cerebral arteries. Isolated mononuclear cells from rabbit peripheral blood were cultured in endothelial growth medium (EGM‐2) for 7 days, yielding EPCs. Rabbits were injected intracisternally with either autologous EPCs (1x10 5 to 5x10 5 cells) or vehicle (EGM‐2) as control. After 24 hours, basilar arteries were isolated, vasomotor function was studied in organ chambers, and protein expression in the basilar arteries quantified by Western blot analysis. In rabbits treated with EPCs, contractions to either potassium chloride (80 mmol/L) or histamine (10 −9 to 10 −5 mol/L) were not affected. However, endothelium‐dependent relaxations to acetylcholine (10 −9 to 10 −5 mol/L) and endothelium‐independent relaxations to a NO donor, DEA‐NONOate (10 −9 to 10 −5 mol/L) were enhanced in basilar arteries from EPC‐treated rabbits. Western blot studies demonstrated increased expressions of cyclooxygenase‐2 and prostacyclin synthase (PGI 2 synthase) only in arteries exposed to EPCs. Results of the present study suggest that in vivo administration of EPCs could augment vasodilatation of the cerebral arteries by increasing vascular production of prostacyclin. Funded by NIH HL66958.

Erythropoietin (EPO) fosters tissue oxygenation by stimulating erythropoiesis. More recently, EPO is recognized as a tissue protective cytokine. In this study, we tested the hypothesis that EPO promotes recovery of injured vascular endothelium. A murine model of wire‐induced injury of common carotid artery was used to examine the therapeutic effect of EPO. Recombinant human EPO (1000 U/kg, s.c., biweekly) was administered for 2 weeks after which vascular reactivity of isolated carotid arteries was studied in vitro under pressurized condition using a video dimension analyzer. Changes in vascular structure were histologically assessed. Injured arteries exhibited impairment in endothelium‐dependent relaxations to acetylcholine and increased thickness of the medial cross sectional area (P<0.05; n=5–7). EPO normalized the vasorelaxant response of injured arteries to acetylcholine and prevented the increase in medial cross sectional area (30±1 x 10 ‐3 mm 2 ; P<0.05 vs. without EPO: 37±3 x 10 ‐3 mm 2 ; n=6). The number of circulating endothelial progenitor cells (EPCs), defined as CD34 + /Flk‐1 + cells in the peripheral blood, was significantly reduced 2 weeks after vascular injury (0.016±0.005%; P<0.05 vs. non‐injured control: 0.09±0.01%), but this was entirely restored by EPO (0.212±0.08%; P<0.05; n=5). Our results indicate that EPO accelerates recovery of the injured carotid artery endothelium, and such salutary effects of EPO may reflect augmentation in circulating EPCs induced by EPO.

The present study is aimed at investigating the contribution of endothelial nitric oxide synthase (eNOS) to erythropoietin (EPO)‐induced mobilization of stem (c‐kit + ,sca‐1 + ,lin‐1 − ; KSL) cells and hematopoietic progenitor (CD34 + ) cells. Wild type (WT) and eNOS −/− mice were injected biweekly with recombinant erythropoietin (1000 U/kg, s.c.) for 14 days. The percentage of KSL cells in spleen, bone marrow and peripheral blood were quantitated by flow cytometry (Becton Dickinson) and analyzed by Cell Quest software. The effect of EPO on number of CD34 + cells in peripheral blood was quantitated by ProCount Progenitor Cell Enumeration Kit (BD Biosciences). Treatment of eNOS −/− mice with EPO increased the percentage of KSL cells by two folds only in bone marrow and spleen, while the percentage of KSL cells in peripheral blood remained unchanged. Treatment with EPO significantly increased the number of CD34 + cells in blood of WT mice (2.75 ± 0.30 cells in WT, 38.39 ± 10.35 in WT+EPO, n=5, P<0.01). However, the number of CD34 + cells remained unchanged in blood of eNOS −/− mice (2.51 ± 1.31 in eNOS −/− , 4.65 ± 1.40 in eNOS −/− + EPO, n=5, P=ns). Results of the present study suggest that eNOS plays an important role in the mobilization of hematopoietic stem/progenitor cells by erythropoietin.

Aging and other cardiovascular risk factors are associated with increased incidence of Alzheimer's disease (AD). A common feature of cardiovascular risk factors is decreased endothelial nitric oxide (NO). We studied the effect of a loss of endothelial NO on amyloid precursor protein (APP) related phenotype in aged (14–15 month) endothelial nitric oxide synthase deficient (eNOS −/− ) mice. APP, β‐site APP cleaving enzyme 1, and amyloid beta (Aβ) levels were significantly higher in the brains of aged eNOS −/− mice as compared to aged wild type mice. APP and Aβ 1–40 were increased in hippocampal tissue of eNOS −/− mice as compared to wild type mice. Aged eNOS −/− mice displayed an increased inflammatory phenotype as compared to aged wild type mice. Importantly, aged eNOS −/− mice performed worse in a radial arm maze test of spatial learning and memory as compared to aged wild type mice. These data suggest that chronic loss of endothelial NO may be an important contributor to both Aβ related pathology and cognitive decline. Research was supported by NIH grants HL‐111062 and HL‐91867, the Mayo Alzheimer's Disease Research Center (Z.S.K.), AHA scientist development grant (0835436N, A.V.S.), AHA postdoctoral fellowship 12POST8550003 (S.A.A.), Clinical Pharmacology Training Grant (T32 GM08685) (Trainee S.A.A.), the Samuel Johnson Foundation for Genomics of Addiction Program at Mayo Clinic (D.‐S.C.) and the Mayo Foundation.

Cilostazol is an inhibitor of phosphodiesterase type III that possesses both antiplatelet and vasodilator effects. The present study aimed to determine the vasodilator effect of cilostazol in eNOS deficient (eNOS −/− ) mice. Mouse aorta was contracted with sub‐maximal concentration of phenylephrine and concentration‐dependent relaxations to cilostazol (10 −8 –10 −5 mol/L) were obtained in aortic rings, isolated from wild type and eNOS −/− mice. As previously reported, in aortas of wild type mice with intact endothelium, relaxations to cilostazol were significantly augmented as compared to aortas without endothelium. Surprisingly, genetic inactivation of eNOS significantly enhanced relaxations to cilostazol suggesting that chronic loss of nitric oxide may unmask compensatory vasodilator mechanisms significantly enhanced by the treatment with cilostazol. The augmentation of relaxations to cilostazol in aortas of eNOS −/− mice was endothelium independent and not inhibited by indomethacin, a cyclooxygenase inhibitor. Our findings support the view that patients suffering from vascular endothelial dysfunction induced by reduced availability of nitric oxide, may exhibit enhanced vasodilator reactivity to cilostazol.

Impaired endothelial function in cardiovascular disorders is mediated, in part, by uncoupling of eNOS caused by reduced availability of tetrahydrobiopterin (BH 4 ). Endothelial progenitor cells (EPCs), originating from bone marrow, contribute to repair of vascular system by either neovascularization following tissue ischemia or re‐endothelialization after endothelial injury. The contribution of BH 4 to the angiogenic function has not been studied. In the present study, we investigated the role of BH 4 in angiogenesis and mobilization of cells enriched in EPCs in hph‐1 mutant mice, a genetic model of GTP‐cyclohydrolase I deficiency. HPLC analysis revealed that enzymatic activity of GTP‐cyclohydrolase I is reduced by 65% in bone marrow of hph‐1 mice (22±4 pmol neopterin/mg/h; P<0.05 vs. wild‐type mice: 63±4 pmol/mg/h). Basal counts of cells co‐expressing CD34 and VEGFR2 (Flk‐1) were reduced in bone marrow and peripheral blood of hph‐1 mice. Fourteen days after hind limb ischemia, recovery of blood flow was significantly reduced in hph‐1 mice as compared to blood flow in ischemic limb of wild type mice. Our findings suggest that reduced bioavailability of BH 4 impairs mobilization of cells enriched in EPCs. This effect may help to explain impaired angiogenesis in hph‐1 mice.

Mobilization of hematopoietic progenitor cells is predictive of functional recovery in stroke. In the present study, alterations in progenitor cell counts were studied in an experimental model of subarachnoid hemorrhage (SAH)‐induced cerebral vasospasm in rabbits. Injection of autologous arterial blood into cisterna magna resulted in significant vasospasm of the basilar arteries forty eight hours following injection. Concomitantly, the number of hematopoietic progenitor cells (CD34 + cells) and CD34 + /Flk‐1 + cells, believed to be enriched in endothelial progenitor cells (EPCs), were significantly increased in the peripheral blood. The levels of the chemokine stromal cell‐derived factor‐1α (SDF‐1α), known to play a key role in the recruitment and retention of CXCR4 + ‐progenitor cells, significantly increased in the plasma of rabbits subjected to SAH. However, levels of SDF‐1α in the cerebrospinal fluid as well as the content of SDF‐1α in the spastic basilar arteries remained unaffected forty eight hours after SAH. In summary, cerebral vasospasm was accompanied by mobilization of hematopoietic progenitor cells and EPCs. We speculate that the pathogenesis of cerebral vasospasm following SAH may be mediated, in part, by impaired recruitment and homing of CXCR4 + ‐ progenitor cells.

The mechanisms underlying dysfunction of cerebral microvasculature induced by type 1 diabetes (T1D) are not fully understood. We hypothesized that in cerebral microvascular endothelium, α-processing of amyloid precursor protein (APP) is impaired by T1D. In cerebral microvessels derived from streptozotocin (STZ)-induced T1D mice protein levels of APP and its α-processing enzyme, a disintegrin and metalloprotease 10 (ADAM10) were significantly decreased, along with down-regulation of adenylate cyclase 3 (AC3) and enhanced production of thromboxane A 2 (TXA 2 ). In vitro studies in human brain microvascular endothelial cells (BMECs) revealed that knockdown of AC3 significantly suppressed ADAM10 protein levels, and that activation of TXA 2 receptor decreased APP expression. Furthermore, levels of soluble APPα (sAPPα, a product of α-processing of APP) were significantly reduced in hippocampus of T1D mice. In contrast, amyloidogenic processing of APP was not affected by T1D in both cerebral microvessels and hippocampus. Most notably, studies in endothelial specific APP knockout mice established that genetic inactivation of APP in endothelium was sufficient to significantly reduce sAPPα levels in the hippocampus. In aggregate, our findings suggest that T1D impairs non-amyloidogenic processing of APP in cerebral microvessels. This may exert detrimental effect on local concentration of neuroprotective molecule, sAPPα, in the hippocampus.

The exact physiological function of amyloid-β precursor protein (APP) in endothelial cells is unknown. Endothelium-specific APP-deficient (eAPP −/− ) mice were created to gain new insights into the role of APP in the control of vascular endothelial function. Endothelium-dependent relaxations to acetylcholine were significantly impaired in basilar arteries of global APP knockout (APP −/− ) and eAPP −/− mice ( P < 0.05). In contrast, endothelium-independent relaxations to nitric oxide (NO)-donor diethylamine-NONOate were unchanged. Western blot analysis revealed that protein expression of endothelial nitric oxide synthase (eNOS) was significantly downregulated in large cerebral arteries of APP −/− mice and eAPP −/− mice as compared to respective wild-type littermates ( P < 0.05). Furthermore, basal levels of cyclic guanosine monophosphate (cGMP) were also significantly reduced in large cerebral arteries of APP-deficient mice ( P < 0.05). In contrast, protein expression of prostacyclin synthase as well as levels of cyclic adenosine monophosphate (cAMP) was not affected by genetic inactivation of APP in endothelial cells. By using siRNA to knockdown APP in cultured human brain microvascular endothelial cells we also found a significant downregulation of eNOS mRNA and protein expressions in APP-deficient endothelium ( P < 0.05). These findings indicate that under physiological conditions, expression of APP in cerebral vascular endothelium plays an important protective function by maintaining constitutive expression of eNOS .

We tested hypothesis that activation of the prostacyclin (PGI 2 ) receptor (IP receptor) signaling pathway in cerebral microvessels plays an important role in the metabolism of amyloid precursor protein (APP). In human brain microvascular endothelial cells activation of IP receptor with the stable analogue of PGI 2 , iloprost, stimulated expression of amyloid precursor protein and a disintegrin and metalloprotease 10 (ADAM10), resulting in an increased production of the neuroprotective and anticoagulant molecule, soluble APPα (sAPPα). Selective agonist of IP receptor, cicaprost, and adenylyl cyclase activator, forskolin, also enhanced expression of amyloid precursor protein and ADAM10. Notably, in cerebral microvessels of IP receptor knockout mice, protein levels of APP and ADAM10 were reduced. In addition, iloprost increased protein levels of peroxisome proliferator-activated receptor δ (PPARδ) in human brain microvascular endothelial cells. PPARδ-siRNA abolished iloprost-augmented protein expression of ADAM10. In contrast, GW501516 (a selective agonist of PPARδ) upregulated ADAM10 and increased production of sAPPα. Genetic deletion of endothelial PPARδ (ePPARδ −/− ) in mice significantly reduced cerebral microvascular expression of ADAM10 and production of sAPPα. In vivo treatment with GW501516 increased sAPPα content in hippocampus of wild type mice but not in hippocampus of ePPARδ −/− mice. Our findings identified previously unrecognized role of IP-PPARδ signal transduction pathway in the production of sAPPα in cerebral microvasculature.