Effects of miR-7 on Hcy-induced rat cerebral arterial vascular smooth muscle cell proliferation, migration and inflammatory factor expression by targeting MMP-14 to regulate TLR4/NF-κB signaling pathway.

The current research aimed to investigate the effect of miR-7 targeting matrix metalloproteinase 14 (MMP-14) on homocysteine (Hcy)-induced rat cerebral artery vascular smooth muscle cells (VSMCs) proliferation, migration and inflammatory factor expression and its possible mechanism. The expression of miR-7 and MMP-14 in Hcy-induced VSMCs were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot. Methyl Thiazolyl Tetrazolium (MTT) method, Transwell assays and enzyme-linked immunosorbent assay (ELISA) were performed to detect the effect of miR-7 and MMP-14 expression on the proliferation and migration, as well as interleukin 6 (IL-6) and tumor necrosis factor ɑ (TNF-ɑ) expression of Hcy-induced VSMCs. The interaction between miR-7 and MMP-14 was detected by dual-luciferase reporter gene assay. Western blot was applied to analyse the effects of miR-7 and MMP-14 expression on the Toll-like receptor (TLR4)/nuclear transcription factor-KB (NF-κB) signaling pathway. The results showed that after induced by Hcy, the expression of miR-7 in VSMCs was significantly reduced, the expression of MMP-14 was significantly increased, and the cell viability, the number of migrating cells, IL-6 and TNF-ɑ expression were significantly increased (P<0.05). After overexpression of miR-7, the viability, migration cell numbers, IL-6 and TNF-ɑ expression of Hcy-induced VSMCs were significantly reduced (P<0.05). miR-7 directly binds to MMP-14 and negatively regulates the expression of MMP-14. After overexpression of miR-7, the levels of TLR4 and p-NF-κB p65 in VSMCs were significantly reduced (P<0.05); overexpression of MMP-14 could reduce the effect of miR-7 overexpression on TLR4 and p-NF-κB p65 expression in VSMCs (P<0.05). Overexpression of MMP-14 and/or activation of the TLR4/NF-κB signaling pathway could reverse the effect of miR-7 overexpression on the proliferation, migration and IL-6 and TNF-ɑ expression of Hcy-induced VSMCs (P<0.05). It is concluded that miR-7 can inhibit Hcy-induced rat cerebral artery VSMCs proliferation, migration, and inflammatory factor expression by targeting the regulation of MMP-14 expression and inhibiting the activation of the TLR4/NF-κB signaling pathway.

Regulation of the cerebrovascular smooth muscle cell phenotype by mitochondrial oxidative injury and endoplasmic reticulum stress in simulated microgravity rats via the PERK-eIF2α-ATF4-CHOP pathway.

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model.

Melatonin activates BKCa channels in cerebral artery myocytes via both direct and MT receptor/PKC-mediated pathway.

The pineal hormone melatonin is a neuroendocrine hormone with high membrane permeability that is involved in regulation of circadian rhythm of several biological functions. Large-conductance Ca2+-activated K+ (BKCa) channels are abundantly expressed in vascular smooth muscle cells and play an important role in vascular tone regulation. We investigated the mechanisms through which myocyte BKCa channels mediate effects of melatonin on cerebral arteries (CAs). Arterial contractility measurements showed that melatonin alone did not change vascular tone in CAs; however, it induced concentration-dependent vasodilation of phenylephrine-induced contraction in CAs. In the presence of the potent endothelial oxide synthase inhibitor, Nω-nitro-L-arginine methyl ester, melatonin-elicited relaxation was significantly inhibited by iberiotoxin (BKCa channel blocker). Melatonin significantly increased BKCa currents but not voltage-gated K+ (KV) currents in whole-cell recordings. Melatonin decreased the amplitude of Ca2+ sparks and spontaneous transient outward currents (STOCs), however, a significant increase in open probability of BKCa channels was observed in both inside-out and cell-attached patch-clamp recordings. This melatonin-induced enhancement of BKCa channel activity was significantly suppressed by luzindole (melatonin MT1/MT2 receptor inhibitor), U73122 (phospholipase C (PLC) inhibitor), and Ro31-8220 (protein kinase C (PKC) inhibitor). Melatonin had no significant effects on sarcoplasmic reticulum release of Ca2+. These findings indicate that melatonin-induced vasorelaxation of CAs is partially attributable to direct (passing through the cell membrane) and indirect (via melatonin MT1/MT2 receptors-PLC-PKC pathway) activation of BKCa channels on CA myocytes.

The G protein-coupled estrogen receptor agonist, G-1, attenuates BK channel activation in cerebral arterial smooth muscle cells.

The G protein-coupled estrogen receptor (GPER) is a significant modulator of arterial contractility and blood flow. The GPER-specific activator, G-1, has been widely used to characterize GPER function in a variety of tissue types. Large conductance, calcium (Ca2+)-activated K+ (BK) channels are sensitive to 17ß-estradiol (17ß-E2) and estrogenic compounds (e.g., tamoxifen, ICI 182 780) that target estrogen receptors. The purpose of this study was to investigate the effects of G-1 on BK channel activation and function in cerebral arterial myocytes. Inside-out and perforated patch clamp were utilized to assess the effects of G-1 (50 nmol·L-1-5 µmol·L-1) on BK channel activation and currents in cerebral arterial myocytes. Pressurized artery myography was used to investigate the effects of G-1 on vasodilatory response and BK channel function of cerebral resistance size arteries. G-1 reduced BK channel activation in cerebral arterial myocytes through elevations in BK channel mean close times. Depressed BK channel activation following G-1 application resulted in attenuated physiological BK currents (transient BK currents). G-1 elicited vasodilation, but reduced BK channel function, in pressurized, endothelium-denuded cerebral arteries. These data suggest that G-1 directly suppresses BK channel activation and currents in cerebral arterial myocytes, BK channels being critically important in the regulation of myocyte membrane potential and arterial contractility. Thus, GPER-mediated vasodilation using G-1 to activate the receptor may underestimate the physiological function and relevance of GPER in the cardiovascular system.

The 15-LO-1/15-HETE system promotes angiogenesis by upregulating VEGF in ischemic brains.

OBJECTIVES: Angiogenesis promotes neurobehavioral recovery after cerebral ischemic stroke. 15(S)-hydroxyeicosatetraenoic acid (15-HETE) is one of the major metabolites of arachidonic acid by 15-lipoxygenase (15-LO) and stimulates the production of vascular endothelial growth factor (VEGF), thus, inducing autocrine-mediated angiogenesis. The present study aimed to investigate the role of 15-LO/15-HETE system on VEGF expression and angiogenesis in brain ischemia. METHODS: Rat cerebral arterial vascular endothelial cells were used to set up a cell injury model of oxygen-glucose deprivation and reoxygenation (OGD/R), mimicking a condition of brain ischemia. A mouse model of middle cerebral artery occlusion (MCAO) was established. RESULTS: Oxygen-glucose deprivation increased cellular expression of 15-LO-1 and VEGF. Transfection of 15-LO-1 siRNA depleted cells of 15-LO-1, and sequentially induced downregulation of VEGF expression; while, incubation of 15-HETE increased the expression of VEGF. Incubation of 15-HETE attenuated the reduction in cell viability induced by oxygen-glucose deprivation, and promoted cell migration, while transfection of 15-LO-1 siRNA showed an opposite effect. In animal experiments, the density of microvessels in hypoxic regions of brains was significantly increased after MCAO, while intracerebroventricular delivery of 15-LO-1 siRNA significantly reduced the density of microvessels, and downregulates VEGF expression. DISCUSSION: The results indicate that the 15-LO-1/15-HETE system promotes angiogenesis in ischemic brains by upregulation of VEGF, representing a potential target for improving neurobehavioral recovery after cerebral ischemic stroke.

Epidermal growth factor-like repeats of tenascin-C-induced constriction of cerebral arteries via activation of epidermal growth factor receptors in rats.

Tenascin-C (TNC), one of matricellular proteins, has been suggested to be involved in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. However, the mechanisms of how TNC constricts cerebral arteries remain unclear. The aim of this study was to examine if epidermal growth factor (EGF)-like repeats of TNC is involved in TNC-induced constriction of cerebral arteries in rats via EGF receptor (EGFR) activation. Two dosages of recombinant TNC (r-TNC) consisting of the EGF-like repeats was administered intracisternally to healthy rats, and its vasoconstrictor effects were evaluated by neurobehavioral tests and India-ink angiography at 24, 48, and 72 hours after the administration. Western blotting and immunohistochemistry were performed to explore the underlying mechanisms on constricted cerebral arteries after 24 hours. The effects of a selective EGFR tyrosine kinase inhibitor (AG1478) on r-TNC-induced vasoconstriction were evaluated by neurobehavioral tests, India-ink angiography and immunohistochemistry at 24 hours after the administration. A higher dosage of r-TNC induced cerebral arterial constriction more severely, which continued for 48 hours. The effects were associated with the activation of EGFR and extracellular signal-regulated kinase (ERK)1/2 in the smooth muscle cell layer of the constricted cerebral artery, while c-Jun N-terminal kinase and p38 were not activated. AG1478 blocked r-TNC-induced vasoconstrictive effects, as well as activation of EGFR and ERK1/2. These findings demonstrate that TNC induces constriction of cerebral arteries via activation of EGFR and ERK1/2.

Role of nucleotide-binding oligomerization domain 1 (NOD1) in pericyte-mediated vascular inflammation.

We have recently described the response of human brain pericytes to lipopolysaccharide (LPS) through toll-like receptor 4 (TLR4). However, Gram-negative pathogen-associated molecular patterns include not only LPS but also peptidoglycan (PGN). Given that the presence of co-purified PGN in the LPS preparation previously used could not be ruled out, we decided to analyse the expression of the intracellular PGN receptors NOD1 and NOD2 in HBP and compare the responses to their cognate agonists and ultrapure LPS. Our findings show for the first time that NOD1 is expressed in pericytes, whereas NOD2 expression is barely detectable. The NOD1 agonist C12-iE-DAP induced IL6 and IL8 gene expression by pericytes as well as release of cytokines into culture supernatant. Moreover, we demonstrated the synergistic effects of NOD1 and TLR4 agonists on the induction of IL8. Using NOD1 silencing in HBP, we showed a requirement for C12-iE-DAP-dependent signalling. Finally, we could discriminate NOD1 and TLR4 pathways in pericytes by pharmacological targeting of RIPK2, a kinase involved in NOD1 but not in TLR4 signalling cascade. p38 MAPK and NF-κB appear to be downstream mediators in the NOD1 pathway. In summary, these results indicate that pericytes can sense Gram-negative bacterial products by both NOD1 and TLR4 receptors, acting through distinct pathways. This provides new insight about how brain pericytes participate in the inflammatory response and may have implications for disease management.

Molecular identification of P2X receptors in vascular smooth muscle cells from rat anterior, posterior, and basilar arteries.

BACKGROUND: Purinergic P2X receptors in vascular smooth muscle cells (VSMCs) play an important role in physiological stimulatory responses to the extracellularly released ATP. The aim of this work was to identify molecular P2X receptor subunits in VSMCs isolated from rat anterior, posterior and basilar arteries using a number of contemporary laboratory techniques. METHODS: P2X mediated ionic currents were recorded using amphotericin B perforated patch clamp method. Gene expression analysis was performed using RT-PCR in manually collected VSMCs. The expression of proteins was confirmed by fluorescent immunocytochemistry. RESULTS: Under voltage clamp conditions VSMCs stimulated by application of 10 µmol/l selective P2X receptor agonist αß-meATP, the biphasic currents consisting of rapidly rising rapidly desensitizing and slowly desensitizing components were observed in freshly isolated myocytes from all three arteries. Using RT-PCR, the expression of genes encoding only P2X1 and P2X4 receptor subunits was detected in preparations from all three arteries. The expression of corresponding P2X1 and P2X4 receptor subunit proteins was confirmed in isolated VSMCs. CONCLUSIONS: Our work therefore identified that in major arteries of rat cerebral circulation VSMCs express only P2X1 and P2X4 receptors subunits. We can propose that these P2X receptor subunits participate in functional P2X receptor structures mediating ATP-evoked stimulatory responses in cerebral vascular myocytes in vivo.

Knockdown of angiopoietin like-2 protects against angiotensin II-induced cerebral endothelial dysfunction in mice.

Angiopoietin like-2 (angptl2) is a circulating pro-inflammatory and pro-oxidative protein, but its role in regulating cerebral endothelial function remains unknown. We hypothesized that in mice knockdown (KD) of angptl2, cerebral endothelial function would be protected against ANG II-induced damage. Subcutaneous infusion of ANG II (200 ng·kg(-1)·min(-1), n = 15) or saline (n = 15) was performed in 20-wk-old angptl2 KD mice and wild-type (WT) littermates for 14 days. In saline-treated KD and WT mice, the amplitude and the sensitivity of ACh-induced dilations of isolated cerebral arteries were similar. However, while endothelial nitric oxide (NO) synthase (eNOS)-derived O2 (-)/H2O2 contributed to dilation in WT mice, eNOS-derived NO (P < 0.05) was involved in KD mice. ANG II induced cerebral endothelial dysfunction only in WT mice (P < 0.05), which was reversed (P < 0.05) by either N-acetyl-l-cysteine, apocynin, gp91ds-tat, or indomethacin, suggesting the contribution of reactive oxygen species from Nox2 and Cox-derived contractile factors. In KD mice treated with ANG II, endothelial function was preserved, likely via Nox-derived H2O2, sensitive to apocynin and PEG-catalase (P < 0.05), but not to gp91ds-tat. In the aorta, relaxation similarly and essentially depended on NO; endothelial function was maintained after ANG II infusion in all groups, but apocynin significantly reduced aortic relaxation in KD mice (P < 0.05). Protein expression levels of Nox1/2 in cerebral arteries were similar among all groups, but that of Nox4 was greater (P < 0.05) in saline-treated KD mice. In conclusion, knockdown of angptl2 may be protective against ANG II-induced cerebral endothelial dysfunction; it favors the production of NO, likely increasing endothelial cell resistance to stress, and permits the expression of an alternative vasodilatory Nox pathway.

The vascular stem cell niches and their significance in the brain.

AIM: This cytohistopathological study was performed for a better knowledge of phenotypes derived from pluripotent stem cells, as well as for precise location of stem cells within the vascular niche in the brain. METHODS: We used light and transmission electron microscopy to demonstrate the presence of stem cells in the vascular wall of microvessels in the cerebral and cerebellar cortex, pia mater (considered by us a cordocytic-vascular tissue), adventitia of larger cortical arteries and veins, and around vessels. We investigated multiple vascular segments and brain tissue in a variety of clinical cases, such as cerebral tumors, cerebrovascular malformations, thromboses in the carotid system, and direct laceration. RESULTS: Our morphological and ultrastructural observations pointed out many changing phenotypes, as well as cell interrelationships within the vascular niche, both for repair processes when cordocytes cooperate with mesenchymal stem cells, and pathological processes such as atherogenesis, tumorigenesis, and neurotrauma. Our results underlie the important roles of cordocytes in their interrelations with precursor/stem cells in the arterial adventitia. CONCLUSION: The cells derived from pluripotent stem cells along different lineages have had different phenotypes as they derived from hematopoietic stem cells or mesenchymal stem cells, with or without epigenetic disregulations or depending on different microenvironments. Cell interactions, phenotypes, and underlying mechanisms, as well as biological responses to different small molecules or compounds, remain to be determined by future molecular insights within the vascular niche.

Pericytes and adaptive angioplasticity: the role of tumor necrosis factor-like weak inducer of apoptosis (TWEAK).

The TNF superfamily member TWEAK has emerged as a pleiotropic cytokine that regulates many cellular functions that include immune/inflammatory activity, angiogenesis, cell proliferation, and fate. TWEAK through its inducible receptor, FGF-inducible molecule 14 (Fn14), can induce both beneficial and deleterious activity that has a profound effect on cell survival. Thus it is highly likely that TWEAK and Fn14 expressed by cells of the neurovascular unit help regulate and maintain vascular and tissue homeostasis. In this chapter we discuss the expression of TWEAK and Fn14 signaling in the cerebral microvascular pericyte. Pericytes are a highly enigmatic population of microvascular cells that are important in regulatory pathways that modulate physiological angiogenesis in response to chronic mild hypoxic stress. A brief introduction will identify the microvascular pericyte. A more detailed discussion of pericyte TWEAK signaling during adaptive angioplasticity will follow.

Protein kinase A regulation of T-type Ca2+ channels in rat cerebral arterial smooth muscle.

Recent investigations have identified that T-type Ca(2+) channels (CaV3.x) are expressed in rat cerebral arterial smooth muscle. In the study reported here, we isolated the T-type conductance, differentiated the current into the CaV3.1/CaV3.2 subtypes and determined whether they are subject to protein kinase regulation. Using patch clamp electrophysiology, whole-cell Ba(2+) current was monitored and initially subdivided into nifedipine-sensitive and -insensitive components. The latter conductance was abolished by T-type Ca(2+) channel blockers and was faster with leftward shifted activation/inactivation properties, reminiscent of a T-type channel. Approximately 60% of this T-type conductance was blocked by 50 µM Ni(2+), a concentration that selectively interferes with CaV3.2 channels. Subsequent work revealed that the whole-cell T-type conductance was subject to protein kinase A (PKA) modulation. Specifically, positive PKA modulators (db-cAMP, forskolin, isoproterenol) suppressed T-type currents and evoked a hyperpolarized shift in steady-state inactivation. Blocking PKA (with KT5720) masked this suppression without altering the basal T-type conductance. A similar effect was observed with stHt31, a peptide inhibitor of A-kinase anchoring proteins. A final set of experiments revealed that PKA-induced suppression targeted the CaV3.2 subtype. In summary, this study revealed that a T-type Ca(2+) channel conductance can be isolated in arterial smooth muscle, and differentiated into CaV3.1 and CaV3.2 components. It also showed that vasodilatory signaling cascades inhibit this conductance by targeting CaV3.2. Such targeting would impact Ca(2+) dynamics and consequent tone regulation in the cerebral circulation.

Distinct activity of BK channel ß1-subunit in cerebral and pulmonary artery smooth muscle cells.

This study was designed to test a hypothesis that the functional activity of big-conductance, Ca(2+)-activated K(+) (BK) channels is different in cerebral and pulmonary artery smooth muscle cells (CASMCs and PASMCs). Using patch-clamp recordings, we found that the activity of whole cell and single BK channels were significantly higher in CASMCs than in PASMCs. The voltage and Ca(2+) sensitivity of BK channels were greater in CASMCs than in PASMCs. Targeted gene knockout of ß(1)-subunits significantly reduced BK currents in CASMCs but had no effect in PASMCs. Western blotting experiments revealed that BK channel α-subunit protein expression level was comparable in CASMCs and PASMCs; however, ß(1)-subunit protein expression level was higher in CASMCs than in PASMCs. Inhibition of BK channels by the specific blocker iberiotoxin enhanced norepinephrine-induced increase in intracellular calcium concentration in CASMCs but not in PASMCs. Systemic artery blood pressure was elevated in ß(1)(-/-) mice. In contrast, pulmonary artery blood pressure was normal in ß(1)(-/-) mice. These findings provide the first evidence that the activity of BK channels is higher in cerebral than in PASMCs. This heterogeneity is primarily determined by the differential ß(1)-subunit function and contributes to diverse cellular responses in these two distinct types of cells.

Distribution of 5-ht(1E) receptors in the mammalian brain and cerebral vasculature: an immunohistochemical and pharmacological study.

BACKGROUND AND PURPOSE: The 5-ht(1E) receptor is highly expressed in the human brain and its structure is conserved in humans, suggesting an important physiological role for 5-ht(1E) receptors. However, neither the function nor the distribution of this receptor has been characterized in the mammalian brain. EXPERIMENTAL APPROACH: Rats and mice lack the 5-ht(1E) receptor gene; thus, we used guinea pig brain tissue and immunofluorescent staining techniques to provide the first specific localization of 5-ht(1E) receptors in the mammalian brain. KEY RESULTS: High levels of 5-ht(1E) receptors are detected in olfactory bulb glomeruli as well as the molecular layer of the dentate gyrus (DG). In DG membranes, BRL54443, a 5-ht(1E) /5-HT(1F) agonist, selectively stimulated 5-ht(1E) receptors and potently inhibited forskolin-dependent cAMP production (IC50 = 14 nM). The staining pattern of 5-ht(1E) receptors in brain tissue suggests that this receptor is expressed predominantly in neurons rather than in glia. Additionally, 5-ht(1E) receptors were detected in the adventitial layer of cerebral arteries but not in the microvasculature, venous tissue or other brain arteries. CONCLUSIONS AND IMPLICATIONS: These observations should help to predict clinical effects of 5-ht(1E) -selective drugs. For example, the stimulation of 5-ht(1E) receptors and subsequent inhibition of adenylate cyclase activity in the DG suggests that 5-ht(1E) receptors may mediate regulation of hippocampal activity by 5-HT, making it a possible drug target for the treatment of neuropsychiatric disorders characterized by memory deficits (such as Alzheimer's disease) or as a target for the treatment of temporal lobe epilepsy.

ASIC-like currents in freshly isolated cerebral artery smooth muscle cells are inhibited by endogenous oxidase activity.

BACKGROUND/AIMS: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. METHODS: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. RESULTS: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 µM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. CONCLUSION: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase.

Isolation and perivascular localization of mesenchymal stem cells from mouse brain.

BACKGROUND: Although originally isolated from the bone marrow, mesenchymal stem cells (MSCs) have recently been detected in other tissues. However, little is known about MSCs in the brain. OBJECTIVE: To determine the extent to which cells with the features of MSCs exist in normal brain tissue and to determine the location of these cells in the brain. METHODS: Single-cell suspensions from mouse brains were cultured according to the same methods used for culturing bone marrow-derived MSCs (BM-MSCs). These brain-derived cells were analyzed by fluorescence-activated cell sorting for surface markers associated with BM-MSCs (stem cell antigen 1 [Sca-1+], CD9+, CD45-, CD11b-, and CD31-). Brain-derived cells were exposed to mesenchymal differentiation conditions. To determine the locations of these cells within the brain, sections of normal brains were analyzed by immunostaining for Sca-1, CD31, and nerve/glial antigen 2. RESULTS: Cells morphologically similar to mouse BM-MSCs were identified and called brain-derived MSCs (Br-MSCs). Fluorescence-activated cell sorting indicated that the isolated cells had a surface marker profile similar to BM-MSCs, ie, Sca-1V+, CD9+, CD45-, and CD11b-. Like BM-MSCs, Br-MSCs were capable of differentiation into adipocytes, osteocytes, and chondrocytes. Immunostaining indicated that Sca-1+ Br-MSCs are located around blood vessels and may represent progenitor cells that serve as a source of mesenchymal elements (eg, pericytes) within the brain. CONCLUSION: Our results indicate that cells similar to BM-MSCs exist in the brain. These Br-MSCs appear to be located within the vascular niche and may provide the mesenchymal elements of this niche. Because MSCs may be part of the cellular response to tissue injury, Br-MSCs may represent targets in the therapy of pathological processes such as stroke, trauma, and tumorigenesis.

Adenosine can mediate its actions through generation of reactive oxygen species.

Adenosine is an important cerebral vasodilator, but mediating mechanisms are not understood. We investigated the expression of adenosine receptor subtypes in isolated cerebral arterial muscle cells (CAMCs), and their role in adenosine-induced superoxide (O(2)(-)) generation and reduction in cerebral arterial tone. Reverse transcriptase-PCR, western blotting, and immunofluorescence studies have shown that CAMCs express transcript and protein for A1, A(2A), A(2B), and A(3) adenosine receptors. Stimulation of CAMCs with adenosine or the A(2A) agonist CGS-21680 increased the generation of O(2)(-) that was attenuated by the inhibition of A(2A) and A(2B) adenosine receptor subtypes, or by the peptide inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase gp91ds-tat, or by the mitochondria uncoupler 2,4-dinitrophenol. Application of adenosine or CGS-21680 dilated pressure-constricted cerebral arterial segments that were prevented by the antioxidants superoxide dismutase (SOD) conjugated to polyethylene glycol (PEG) and PEG-catalase or by the A(2B) adenosine receptor antagonist MRS-1754, or by the mixed A(2A) and A(2B) antagonist ZM-241385. Antagonism of the A(2A) and A(2B) adenosine receptors had no effect on cerebral vasodilatation induced by nifedipine. These findings indicate that adenosine reduces pressure-induced cerebral arterial tone through stimulation of A(2A) and A(2B) adenosine receptors and generation of O(2)(-) from NADPH oxidase and mitochondrial sources. This signaling pathway could be one of the mediators of the cerebral vasodilatory actions of adenosine.

Adenosine analogue-oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIalpha).

INTRODUCTION: Type I cGMP-dependent protein kinase (PKGIalpha) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIalpha is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIalpha would lead to advances in the treatment of a variety of cardiovascular diseases. AIM: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIalpha to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. RESULTS: Structure-activity profiling of ARCs with PKGIalpha in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC(50) values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1alpha-induced vasodilation.

Structure and functional loading of cerebral arterial smooth myocyte population.

The cerebral arterial smooth myocytes of dogs with experimental hypertension (n=12), its correction (n=10), and of intact dogs (n=8) were studied. The structure of myocyte population changed under conditions of vascular wall hyperfunction: the size of the cytoplasm and nuclei, DNA content in the nuclei, mitotic activity, and percentage of binuclear forms increased. Thickening of the tunica media in intraorgan arteries was caused by such cytological mechanisms as leiomyocyte hyperplasia and hypertrophy. The cells shrank after elimination of hypertension, while vascular polyploidy persisted.

Therapeutic benefits of angiogenetic gene-modified human mesenchymal stem cells after cerebral ischemia.

Intravenous transplantation of human mesenchymal stem cells (hMSCs) expanded from adult bone marrow ameliorates functional deficits in rat cerebral infarction models. Several hypotheses to account for the therapeutic mechanisms have been suggested, but angiogenesis is thought to be of critical importance. Recently, we have reported the therapeutic benefits of hMSCs which have been transfected with the angiopoietin-1 gene in a rat permanent middle cerebral artery occlusion (MCAO) model. To potentially enhance the therapeutic effects of angiopoietin-1 gene-modified hMSC (Ang-hMSC), we transfected hMSCs with the angiopoietin-1 gene and the VEGF gene, and investigated whether the combination of Ang-1 and VEGF gene-modified hMSCs (Ang-VEGF-hMSC) contribute to functional recovery in a rat MCAO model. We induced MCAO using intraluminal vascular occlusion, and hMSCs, Ang-hMSCs, VEGF-hMSCs or Ang-VEGF-hMSCs were intravenously infused 6 h later. MRI and behavioral analyses revealed that rats receiving Ang-VEGF-hMSCs showed the greatest structural-functional recovery as compared to the other groups. These results suggest that intravenous administration of hMSCs transfected with the angiopoietin-1 and VEGF gene using a fiber-mutant adenovirus vector may represent a new strategy for the treatment of ischemia.