Characterization of microvascular cell cultures from normotensive and hypertensive rat brains: pericyte-endothelial cell interactions in vitro

We used specific markers and fluorescence microscopy to identify and characterize cerebrovascular cells. Cultures were derived from brain microvessels isolated from normotensive (Wistar Kyoto, WKY) and spontaneously hypertensive (SHR) rat brains prior to, coincident with and following the onset of chronic hypertension. Endothelial cells were characterized using di-acyl LDL and non-muscle isoactin-specific antibodies. Cerebrovascular pericytes were identified with the anti-muscle and non-muscle actin antibody staining. Using this combination of cell culture and fluorescence localization, we have been able to demonstrate that brain pericytes are tightly associated with the endothelial cells of the hypertensive-prone and hypertensive cell cultures, but not with the normotensive endothelial cultures. While the endothelial-pericyte ratio in the hypertensive-prone microvascular cultures was between 5:1 and 10:1, the number of pericytes associated with the hypertensive rat brain cultures increased two to five times (2:1-1:1). Muscle and non-muscle actin antibody staining localized the spindle-shaped pericytes of the hypertensive microvascular colonies. Pericytes were found overlaying and encircling the endothelial cells. Normotensive pericytes were not endothelial-associated. Whereas the hypertensive pericyte is devoid of stress fibers, the normotensive pericyte is a larger, spread-out cell possessing numerous stress fibers rich in muscle and non-muscle actin. These results provide the first evidence that the etiology and inception of cerebrovascular disease may be pericyte-related and suggest that pericyte contraction could play a pivotal role in regulating the flow of blood within the brain microcirculation.     

Microvascular pericytes contain muscle and nonmuscle actins

We have affinity-fractionated rabbit antiactin immunoglobulins (IgG) into classes that bind preferentially to either muscle or nonmuscle actins. The pools of muscle- and nonmuscle-specific actin antibodies were used in conjunction with fluorescence microscopy to characterize the actin in vascular pericytes, endothelial cells (EC), and smooth muscle cells (SMC) in vitro and in situ. Nonmuscle-specific antiactin IgG stained the stress fibers of cultured EC and pericytes but did not stain the stress fibers of cultured SMC, although the cortical cytoplasm associated with the plasma membrane of SMC did react with nonmuscle-specific antiactin. Whereas the muscle-specific antiactin IgG failed to stain EC stress fibers and only faintly stained their cortical cytoplasm, these antibodies reacted strongly with the fiber bundles of cultured SMC and pericytes. Similar results were obtained in situ. The muscle-specific antiactin reacted strongly with the vascular SMC of arteries and arterioles as well as with the perivascular cells (pericytes) associated with capillaries and post-capillary venules. The non-muscle-specific antiactin stained the endothelium and the pericytes but did not react with SMC. These findings indicate that pericytes in culture and in situ possess both muscle and nonmuscle isoactins and support the hypothesis that the pericyte may represent the capillary and venular correlate of the SMC.     

Functional sorting of actin isoforms in microvascular pericytes

We characterized the form and distribution of muscle and nonmuscle actin within retinal pericytes. Antibodies with demonstrable specificities for the actin isoforms were used in localization and immunoprecipitation experiments to identify those cellular domains that were enriched or deficient in one or several actin isoforms. Living pericyte behavior was monitored with phase-contract video microscopy before fixation to identify those cellular areas that might preferentially be stained with either of the fluorescent antiactins or phallotoxins. Antibody and phallotoxin staining of pericytes revealed that nonmuscle actin is present within membrane ruffles, pseudopods, and stress fibers. In contrast, muscle actin could be convincingly localized in stress fibers, but not within specific motile areas of pericyte cytoplasm. To confirm and quantitatively extend the results obtained by fluorescence microscopy, nonionic and ionic detergents were used to selectively extract the motile or immobilized (stress fiber-containing) regions of biosynthetically labeled pericyte cytoplasm. Immunoprecipitated actins that were present within these discrete cellular domains were subjected to isoelectric focusing in urea-polyacrylamide gels before fluorographic analysis. Scanning laser densitometry of the focused actins could not reveal any detectable alpha-actin within those beta- and gamma-actin-enriched motile regions extracted with nonionic detergents. Moreover, when pericyte stress fibers are completely dissolved by ionic detergent lysis, three actin isoforms can be quantified to be present in a ratio of 1:2.75:3 (alpha:beta:gamma). These biochemical findings on biosynthetically labeled and immunoprecipitated pericyte actins confirm the fluorescent localization studies. While the regulatory events governing this actin sorting are unknown, it seems possible that such events may play important roles in controlling cell shape, adhesion, or the promotion of localized cell spreading.     

Atrial natriuretic peptide and angiotensin II binding sites in cerebral capillaries of spontaneously hypertensive rats

1. We carried out investigations on specific atrial natriuretic peptide (ANP) and angiotensin II (ANG) binding sites in capillaries isolated from the cerebral cortex of spontaneously hypertensive rats (SHR), an animal model of human essential hypertension, and also from Wistar Kyoto rats (WKY). 2. In an equilibrium binding study done in the presence of increasing concentrations of the radiolabeled ligands, the binding of 125I-rat alpha-ANP (1-28) [ANF-(99-126)] (125I-rANP) and 125I-ANG (5-L-isoleucine) (125I-ANG) to the cerebral capillaries was single and of a high affinity. 3. The maximum binding capacity (Bmax) and dissociation constant (Kd) in the 125I-rANP binding of 20-week-old, hypertensive SHR was significantly lower than in age-matched, normotensive WKY. Conversely, a significant increase in the Bmax of 125I-ANG binding of adult SHR was observed, with a significant decrease in the Kd. 4. There was no differences in the Bmax of 125I-rANP and 125I-ANG binding between 4-week-old, prehypertensive SHR and age-matched WKY. However, there was a significant decrease in the Kd of 125I-rANP binding of SHR. 5. As a dramatic change in the binding kinetics of 125I-rANP and 125I-ANG was noted in the cerebral capillaries of adult sustained-hypertensive SHR, the possibility that ANP and ANG play a role in the etiology of dysfunction of the blood-brain barrier complicated with hypertension, by interacting with specific receptors, would have to be considered.     

Increased rhythmicity in hypertensive arterial smooth muscle is linked to transient receptor potential canonical channels

Vasomotion describes oscillations of arterial vascular tone due to synchronized changes of intracellular calcium concentrations. Since increased calcium influx into vascular smooth muscle cells from spontaneously hypertensive rats (SHR) has been associated with variances of transient receptor potential canonical (TRPC) channels, in the present study we tested the hypothesis that increased vasomotion in hypertension is directly linked to increased TRPC expression. Using a small vessel myograph we observed significantly increased norepinephrine‐induced vasomotion in mesenteric arterioles from SHR compared to normotensive Wistar–Kyoto (WKY) rats. Using immunoblottings we obtained significantly increased expression of TRPC1, TRPC3 and TRPC5 in mesenteric arterioles from SHR compared to WKY, whereas TRPC4 and TRPC6 showed no differences. Norepinephrine‐induced vasomotion from SHR was significantly reduced in the presence of verapamil, SKF96365, 2‐aminoethoxydiphenylborane (2‐APB) or gadolinium. Pre‐incubation of mesenteric arterioles with anti‐TRPC1 and anti‐TRPC3 antibodies significantly reduced norepinephrine‐induced vasomotion and calcium influx. Control experiments with pre‐incubation of TRPC antibodies plus their respective antigenic peptide or in the presence of anti‐β‐actin antibodies or random immunoglobulins not related to TRPC channels showed no inhibitory effects of norepinephrine‐induced vasomotion and calcium influx. Administration of candesartan or telmisartan, but not amlodipine to SHR for 16 weeks significantly reduced either the expression of TRPC1, TRPC3 and TRPC5 as well as norepinephrine‐induced vasomotion in mesenteric arterioles. In conclusion we gave experimental evidence that the increased TRPC1, TRPC3 and TRPC5 expression in mesenteric arterioles from SHR causes increased vasomotion in hypertension.     

Pericytes of the brain microvasculature express gamma-glutamyl transpeptidase.

The expression of gamma-glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood-brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary-specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT-protein-A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in porcine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary-adjacent pericytes in addition to the GGT-positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.     

Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats

The brain uptake and brain to blood efflux transport of (14)C-GABA were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using 20 min bilateral in situ brain perfusion in rats anesthetized using urethane. The volume of distribution (Vd) of (14)C-GABA into cerebrospinal fluid (CSF) and brain regions (cortex, diencephalon, cerebellum, and brain stem) was significantly greater in SHR than in the corresponding regions in WKY rats (p<0.05). The estimated Vd value of (14)C-GABA in CSF of SHR was 3.4 fold greater than that in WKY. Also compared to WKY, the Vd of (14)C-GABA into cerebellum and cortex of SHR was 15.3 fold and 19.4 fold greater, respectively. Although the study of blood-brain barrier (BBB) integrity using (3)H-mannitol revealed increased paracellular permeability at the brain capillaries of SHR when compared to WKY rats, this was found to be only partially responsible for the increased (14)C-GABA uptake. The study of brain to blood efflux transport of (14)C-GABA (after loading of brain with (14)C-GABA by vascular perfusion) revealed that the half-time of elimination was significantly shorter in SHR (5.35+/-0.66 min) than in WKY rats (14.83+/-1.94 min), (p<0.001). HPLC analysis revealed that GABA concentrations in brain extracts and CSF of SHR were similar to those in WKY rats (p>0.05). The faster efflux in SHR might be, at least partially, responsible to compensate for increased uptake of this neurotransmitter and to preserve the protective function of BBB towards GABA. The protective function of the BCSFB towards GABA appears to be also preserved, since systemic infusion of GABA within a wide range of administered doses (0.004-5.00 mg/kg) produced an increase in GABA CSF concentration from around 0.5 microM to only 11 microM, and the obtained pattern of CSF GABA concentrations under these conditions did not differ between SHR and WKY rats, as revealed by HPLC.     

Establishment of conditionally immortalized rat retinal pericyte cell lines (TR-rPCT) and their application in a co-culture system using retinal capillary endothelial cell line (TR-iBRB2)

The purpose of this study was to establish and characterize a retinal pericyte cell line from retinal capillaries of transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene (tsA58 Tg rat), and to apply this to the co-culture with a retinal capillary endothelial cell line. The conditionally immortalized rat retinal pericyte cell lines (TR-rPCTs), which express a temperature-sensitive large T-antigen, were obtained from two tsA58 Tg rats. These cell lines had a multicellular nodule morphology and reacted positively with von Kossa staining, a marker of calcification. TR-rPCTs cells expressed mRNA of pericyte markers such as rat intercellular adhesion molecule-1, platelet-derived growth factor-receptor beta, angiopoietin-1, and osteopontin. Western blot analysis indicated that alpha-smooth muscle actin (alpha-SMA) was expressed in TR-rPCT3 and 4 cells. In contrast, alpha-SMA was induced by transforming growth factor-beta1 and its enhancement was reduced by basic fibroblast growth factor in TR-rPCT1 and 2 cells. When TR-rPCT1 cells were cultured with a rat retinal endothelial cell line (TR-iBRB2) in a contact co-culture system, the number of TR-iBRB2 cells were significantly reduced in comparison with that of a single culture of TR-iBRB2 cells, suggesting that physical contact between pericytes and retinal endothelial cells is important for the growth of retinal endothelial cells. In conclusion, conditionally immortalized retinal pericyte cell lines were established from tsA58 Tg rats. These cell lines exhibited the properties of retinal pericytes and can be applied in co-culture systems with a retinal capillary endothelial cell line.     

Renal corticosterone 6β-hydroxylase in the spontaneously hypertensive rat

Excess 6β-OH-corticosterone production by family 3A cytochromes P-450 may play a role in genesis of hypertension in the spontaneously hypertensive rat (SHR), by producing a renal defect in Na⁺ excretion. Renal cytochromes P-450 may be a causal factor in this genetic model. Since family 3A P-450 is present in rat kidney (collecting duct), the renal family 3A catalytic (6β-OHase) and immunoreactive activities were compared in SHR and normotensive control (Wistar-Kyoto; WKY) rats. Corticosterone 6β-hydroxulation is markedly higher in SHR than in WKY renal microsomal preparations. Western blot analysis with antibodies to rat and rabbit liver family 3A isoforms demonstrated related proteins. Densitometry revealed greater relative intensity of staining in SHR compared to WKY with both antibodies. Both antibodies inhibited corticosterone 6β-hydroxylation by SHR renal microsomes. Increased renal 6β-OH-corticosterone production by increased renal family 3A cytochromes P-450 may play a role in the blood pressure elevation in SHR.     

Contractile proteins in pericytes at the blood-brain and blood-retinal barriers

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (α-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that α-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to α-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.     

成年SHR动脉平滑肌细胞端粒酶活性和周期蛋白D1的研究

为探索自发性高血压大鼠动脉平滑肌细胞(SMC)增生的机理,采用3H-TdR标记、端粒酶活性以及细胞周期蛋白D1基因RT-PCR检测分别对10周龄SHR、WKY大动脉及其体外分离的SMC进行研究。成年、高血压状态的SHR胸、腹主动脉段端粒酶有高的活性,而同龄、同源WKY大鼠者则没有。从成年SHR腹主动脉段分离的SMC3H-TdR的掺入率比WKY者约提高了43％。成年高血压状态下的SHR腹主动脉SMC细胞周期蛋白D1基因的RT-PCR的产物与WKY者相差不明显。     

Abnormal recruitment of periendothelial cells to lymphatic capillaries in digestive organs of angiopoietin-2-deficient mice

The fine structure of lymphatic capillaries in the digestive organs of angiopoietin-2 (Ang2) knockout mice was studied by using both immunohistochemistry and electron microscopy. The genetic deletion of Ang2 yielded hypoplasia and disorganization of the lymphatic capillaries, with their shapes being irregular, and an aberrant recruitment of vascular periendothelial cells immunopositive for smooth muscle actin to the lymphatic capillaries. The abnormal lymphatic periendothelial cells were considered to be a type of pericyte for the lymphatic capillaries after the deletion of Ang2, because they were ultrastructurally characterized by abundant thin myofilaments in their cytoplasm and long cytoplasmic extensions similar to those shown by blood vascular pericytes. The genetic replacement of Ang2 with Ang1 rescued the defects, viz., the disorganization and disordered structure of the lymphatic capillaries. The present findings suggest that Ang2 serves the morphogenesis of lymphatic capillaries as an agonist for the receptor, Tie2, and that Ang1 can replace Ang2 in guiding lymphatic formation and development. H. Shimoda and M. Witte were supported by an Exchange Visitor Program Grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by Contract 6011 from the Arizona Disease Control Research Commission, respectively.     

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.     

Differential stimulation of growth related metabolism in cultured smooth muscle cells from SHR and WKY rats by combinations of EGF and LDL

We have reported previously that vascular smooth muscle cells from spontaneously hypertensive rats (SHR) were more responsive to epidermal growth factor (EGF) than their normotensive derived Wistar Kyoto (WKY) controls. This differential responsiveness is evident for several cellular processes including activation of S6-kinase, elevation of intracellular pH and stimulation of both phosphoinositide metabolism and DNA synthesis. Quiescent smooth muscle cells exposed to low density lipoprotein (LDL) exhibited a similar differential responsiveness (SHR greater than WKY) in terms of S6-kinase activation, which was time- and dose-dependent (10(-10)-10(7) M), but neither cell type responded appreciably to LDL in terms of a stimulation in [3H]-thymidine incorporation. Exposure of the same cells to EGF and LDL in combination elicited a marked synergistic stimulation in DNA synthesis, the extent of which was greater for SHR than WKY. The sensitivity of both cell types to EGF was increased in the presence of LDL, although cells from hypertensive animals still exhibited their greater (vs. WKY) sensitivity. In both cell types, activation of nuclear protooncogenes c-fos and c-myc by LDL was minimal, whereas oncogene induction by EGF was approximately five-fold greater for SHR-derived cells compared to those from WKY animals. No marked synergistic effect on the time-dependent induction of either entity was observed for cells exposed to EGF and LDL simultaneously, and the response of SHR-cells remained greater than WKY-cells.     

Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin-(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(1-12). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(1-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(1-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(1-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels.     

Changes in Proteomic Features Induced by Insulin on Vascular Smooth Muscle Cells from Spontaneous Hypertensive Rats In Vitro

Hyperinsulinemia is a risk factor in atherosclerosis formation that it stimulated vascular smooth muscle cells (VSMCs) proliferation and migration. To understand the underlying molecular mechanism involved in the processes of cellular response to insulin, VSMCs from Wistar-Kyoto rat (WKY) and spontaneous hypertensive rat (SHR) were isolated and cultured, and its proteome was comparatively analyzed with normal control by two-dimensional gel electrophoresis (2-DE). Results showed that the proliferation of VSMCs from SHR be more sensitive to insulin stimulation than that VSMCs from WKY. The detectable spots ranged from 537 to 608 on the gels in VSMCs of SHR, and 413 ± 31 spots in VSMCs of WKY. The different expressed protein spots in VSMCs of SHR were then isolated and measured by matrix-assisted desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). A total of 18 spots showed a sharp clear spectrum, and 13 spots matched with the known proteins from database. These proteins were mainly involved in cytoskeleton, glycometabolism, and post-translational processes. Among these proteins, OPN and matrix gla protein were up-regulated expression proteins, while α-SM actin was down-regulated. Furthermore, these preliminarily identified proteins confirmed by RT-PCR and western blotting analysis were coincident with the changes in 2-DE check. In addition, the cytoskeleton changes and migration rate of VSMCs from SHR treated by insulin increased significantly. The results showed that insulin plays a crucial role in activating proliferation and migration of VSMCs, by regulating the phenotype switch of VSMCs.     

Dependence of the number of vesicles in vascular smooth muscle cells on the presence of noradrenaline in the extracellular space

Noradrenaline (NA) effect on the number of vesicles in smooth muscle cells was investigated in small mesenteric arteries of spontaneously hypertensive rats (SHR), aged 8 or 12 weeks, and age-matched normotensive Wistar-Kyoto (WKY) rats. The presence of NA in the incubation medium resulted in an increase in the number of vesicles in SHR of both age groups, but not in WKY. The results are discussed in view of the relationship between the vesicles and Ca transport in smooth muscle cells.     

A Novel In Vitro Model to Study Pericytes in the Neurovascular Unit of the Developing Cortex

Cortical function is impaired in various disorders of the central nervous system including Alzheimer’s disease, autism and schizophrenia. Some of these disorders are speculated to be associated with insults in early brain development. Pericytes have been shown to regulate neurovascular integrity in development, health and disease. Hence, precisely controlled mechanisms must have evolved in evolution to operate pericyte proliferation, repair and cell fate within the neurovascular unit (NVU). It is well established that pericyte deficiency leads to NVU injury resulting in cognitive decline and neuroinflammation in cortical layers. However, little is known about the role of pericytes in pathophysiological processes of the developing cortex. Here we introduce an in vitro model that enables to precisely study pericytes in the immature cortex and show that moderate inflammation and hypoxia result in caspase-3 mediated pericyte loss. Using heterozygous EYFP-NG2 mouse mutants we performed live imaging of pericytes for several days in vitro. In addition we show that pericytes maintain their capacity to proliferate which may allow cell-based therapies like reprogramming of pericytes into induced neuronal cells in the presented approach.