Capillary Pericytes Research Articles

Characterization of glycerophosphocholine phosphodiesterase activity and phosphatidylcholine biosynthesis in cultured retinal microcapillary pericytes. Effect of adenosine and endothelin-1.

In pericytes from bovine retina, the enzyme glycerophosphocholine phosphodiesterase, catalyzing the hydrolysis of sn-glycero-3-phosphocholine to glycero-3-phosphate and choline, has been characterized with respect to pH optimum, metal ion dependence, Km, inhibitors, and subcellular localization. In these cells, the natural substrate sn-glycero-3-phosphocholine was present at relatively high concentration (6.4 +/- 1.2 nmol/mg protein), and the EDTA-sensitive phosphodiesterase activity was also found to be markedly high (9.80 +/- 1.5 nmol/min/mg protein) compared to that estimated in liver and brain (1-3 nmol/min/mg protein) or in renal epithelial cell culture (0.27 nmol/min/mg protein). The reaction conditions were in general agreement with those found earlier in brain and other tissues. The majority of the enzyme specific activity was located in the plasma membrane, whereas a minor part was present in the microsomal fraction. The physiological significance of the high catabolic phosphodiesterase activity in these cells may be related to the transfer, followed by deacylation, of lysophosphatidylcholine from the bloodstream to nervous tissue. In addition, capillary pericytes in culture were able to incorporate 3H-choline rapidly into choline-containing soluble phosphorylated intermediates and into phosphatidylcholine. To find a positive and negative effector on phosphatidylcholine formation, adenosine, an important intercellular mediator in the retina in response to alterations in oxygen delivery, and endothelin-1, a potent paracrine mediator present at the blood-brain and blood-retina barrier, were tested. The cells cultured for 1 or 24 h in a medium containing adenosine at concentrations of 10(-6) and 10(-4) M showed significant reduction in 3H-choline incorporation compared to control cultures, whereas endothelin-1, at a concentration of 10 and 100 nM, caused stimulation of phosphatidylcholine biosynthesis. These findings provide evidence that both agonists may modulate phosphatidylcholine metabolism in pericytes.

Ultrastructural changes in the dorsal skin of Wistar-derived hypotrichotic WBN/ILA- Ht rats following UVA-irradiation

Ultrastructual characteristics of the dorsal skin responses to a single irradiation of UVA (1100 kJ/m2) were examined in Wistar-derived hypotichotic WBN/ILA-Ht rats (HtRs). In the epidermis, mitochondrial swelling of some keratinocytes and dissociation of keratinocytes due to intercellular edema developed at 3 hours (h) after irradiation and continued to 48 h. At 6 h, in addition to these changes, necrosis of keratinocytes accompanied with infiltration of neutrophils was also observed in some portions, and epidermal hyperplasia with many keratinocytes showing nucleolar hypertrophy and some mitotic keratinocytes was observed at 48 h. In the dermis, mitochondrial swelling and/or partial cytoplasmic destruction in capillary endothelial cells and edema with inflammatory cell infiltration were observed at and after 3 h, and extravasation of erythrocytes was found in some capillaries at 48 h. Mitochondrial swelling was also frequently found in pericytes and fibroblasts. Inflammatory cells were mainly composed of neutrophils throughout the experimental period. Mild degranulation of mast cells which also showed mitochondrial swelling was observed at and after 3 h, and a close special relationship between mast cells and fibroblasts or neutrophils was sometimes observed. In conclusion, the most prominent change in the dorsal skin of HtRs exposed to UVA was degeneration of capillary endothelial cells, resulting in edema and inflammatory cell infiltration, and the most characteristic cytopathic effect of UVA was mitochondrial swelling, and it was common to keratinocytes, capillary endothelial cells, pericytes, mast cells, and fibroblasts.

Exogenous advanced glycosylation end products induce diabetes-like vascular dysfunction in normal rats: a factor in diabetic retinopathy.

Diabetic retinopathy has been shown to be directly associated with the degree and duration of hyperglycemia, and advanced glycosylation end products (AGEs) have been implicated in this pathological process. The purpose of the experiments reported here was to study the effect of AGE deposition on retinal vascular damage which leads to diabetic retinopathy. Intravenous injection of exogenous AGEs was used to treat wild-type non-diabetic Sprague-Dawley rats. One of the two retinal slides from each animal was treated using immunohistochemical staining to label retinal vascular AGE deposition, the other H&E staining for counting of capillary pericytes. The results were compared with the findings in untreated wild-type and diabetic controls and in rats treated with unmodified rat serum albumin (RSA). After 2 weeks of continuous treatment, AGEs were identified in the retinal vascular tissue of the AGE-RSA-injected group. The average number of retinal capillary pericytes per 10x100 microscope power field was 4.313+/-0.34 (mean +/- SD) in the AGE-RSA-injected group, compared with 5.798+/-0.481 in the control group ( P<0.01). These experiments demonstrate that AGEs, independent of other metabolic factors, can induce vascular change resembling that of diabetic retinopathy.

Sorbitol dehydrogenase overexpression potentiates glucose toxicity to cultured retinal pericytes

The polyol pathway consists of two enzymes, aldose reductase (AR) and sorbitol dehydrogenase (SDH). There is a growing body of evidence to suggest that acceleration of the polyol pathway is implicated in the pathogenesis of diabetic vascular complications. However, a functional role remains to be elucidated for SDH in the development and progression of diabetic retinopathy. In this study, cultured bovine retinal capillary pericytes were used to investigate the effects of SDH overexpression on glucose toxicity. High glucose modestly increased reactive oxygen species (ROS) generation, decreased DNA synthesis, and up-regulated vascular endothelial growth factor (VEGF) mRNA levels in cultured pericytes. SDH overexpression was found to significantly stimulate ROS generation in high glucose-exposed pericytes and subsequently potentiate the cytopathic effects of glucose. Fidarestat, a newly developed AR inhibitor, and N-acetylcysteine, an antioxidant, completely prevented these deleterious effects of SDH overexpression on pericytes. Furthermore, fidarestat administration was found to significantly prevent vascular hyperpermeability, the characteristic changes of the early phase of diabetic retinopathy, in streptozotocin-induced diabetic rats. Our present results suggest that SDH-mediated conversion of sorbitol to fructose and the resultant ROS generation may play an active role in the pathogenesis of diabetic retinopathy. Blockage of sorbitol formation by fidarestat could be a promising therapeutic strategy for the treatment of early phase of diabetic retinopathy.

Selective pericyte degeneration in the retinal capillaries of galactose-fed dogs results from apoptosis linked to aldose reductase-catalyzed galactitol accumulation

Galactose-fed dogs develop retinal capillary changes similar to diabetic retinopathy with pericyte degeneration as the initial lesion. This is followed by the formation of microaneurysms, hemorrhages, and some areas of acellularity. To investigate the mechanisms for selective pericyte degeneration, retinal capillary pericytes and endothelial cells isolated from beagle dog retina were cultured for 2 weeks in Dulbecco's modified Eagle's medium (DMEM) containing 50 mM d-galactose. Apoptosis was detected in pericytes but not endothelial cells by in situ terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick end labelling (TUNEL) staining and the DNA fragmentation assay on agarose gel electrophoresis. This apoptosis was prevented by the addition of the aldose reductase inhibitor AL 1576 to the culture medium containing galactose. Apoptosis was not observed when pericytes were similarly cultured in control DMEM medium. These data support the premise that the selective degeneration of retinal capillary pericytes observed in galactose-fed dogs is linked to increased aldose reductase activity in these cells.

The bovine placenta; a source and target of steroid hormones: observations during the second half of gestation

Apart from estrone-3-sulfate (E1S) the bovine placenta produces progesterone (P4), though the corpus luteum is the major source of P4 responsible for maintaining pregnancy. So far the biological function of placental steroids in cattle is largely unknown. However, since the local availability of free estrone (E1) in the placenta seems to be controlled by sulfatase and sulfotranferase, the hypothesis was developed that placental estrogens and P4 might act as local regulatory factors. To test for such a function placentomes from 150, 220, 240, 270 days (D) pregnant and parturient cows were screened immunohistochemically for progesterone and estrogen receptors (PR, ER). PR were found at all stages in the caruncle in stromal cells and capillary pericytes but only at parturition in arterial walls. Percentage of PR-positive caruncular stromal cells (CSC) increased ( P<0.05) from 51.8±2.6% at D150 to 58.9±1.8% at parturition. ER were detected in CSC, caruncular epithelial (CE) cells and in caruncular capillary pericytes. Mean percentage of ER-positive CSC decreased from 39.0±5.9% in pregnant cows to 17.5±8.3% at parturition ( P<0.05). In CE all cells exhibited positive signals with the exception of those immediately surrounding large primary chorionic villi. Proliferation was assessed immunohistochemically by determining the percentage of Ki67-antigen positive cells. Highest values ( P<0.001) were obtained for CE (58.0–68.3%), followed by the trophoblast (23.3–25.4%), CSC (10.6–45.3%) and the stroma of the chorionic villi (2.9–10.5%). A transient depression of proliferation in CSC between D150–270 ( P<0.05) paralleled local estrogen tissue concentrations. The results suggest that placental estrogens and P4 are important factors controlling caruncular growth, differentiation and function.

Occurrence of Estrogen Receptor α in Bovine Placentomes Throughout Mid and Late Gestation and at Parturition1

The bovine placenta produces estrogens from the first trimester until the end of its life span. However, with the exception of the immediate prepartal and intrapartal phases, in which an involvement of placental estrogens has been suggested for the preparation of parturition, their function has not been elucidated yet. To test for a role of placental estrogens as local factors regulating placental growth and differentiation, placentomes from cows that were pregnant for 150, 220, 240, and 270 days, and parturient cows (3 animals per group) were screened immunohistochemically for the expression of estrogen receptor alpha (ERalpha). Indirect immunoperoxidase staining methods were applied using primary monoclonal antibodies (pmAbs) directed against the C-terminus (AER311, HT277) or the N-terminus (AER314, 1D5) of the ERalpha molecule. Both types of pmAbs identified ERalpha in stromal cells and capillary pericytes of the maternal caruncular septae. Using pmAb 1D5, the mean percentage of ERalpha-positive caruncular stromal cells decreased from 39.0% +/- 5.9% in pregnant cows to 17.5% +/- 8.3% at parturition (P = 0.011). Only pmAb recognizing the C-terminus identified ERalpha in the caruncular epithelium, in which positive reactions were found in all cells, with the exception of areas adjacent to the chorionic plate and to major chorionic villi, where the specific signal gradually faded and occasionally disappeared. No positive reactions were observed in the fetal part of the placentomes. The expression of ERalpha in bovine placentomes was further confirmed by the detection of ERalpha-specific mRNA by reverse transcriptase-polymerase chain reaction and by Western blot analysis. The results suggest a role for placental estrogens as paracrine factors involved in the regulation of placental growth and differentiation.

Three-dimensional wall structure and the innervation of dental pulp blood vessels.

The involvement of neural components in plasma extravasation and blood flow in the dental pulp has been established by pharmacological and physiological studies. We review here the segmental constitution of pulp vessels and the possible involvement of neural components in both the contractility and permeability of the pulp vessels from a morphological viewpoint. Six vascular segments can be identified based on the morphology of peri-endothelial cells, such as smooth muscle cells and pericytes. These are: muscular arterioles, terminal arterioles, precapillary arterioles, capillaries, postcapillary venules, and collecting or muscular venules. The perivascular nerve forms a mesh with numerous terminal varicosities, some of which attach directly to arteriolar smooth muscle cells. This mesh can be seen by scanning electron microscopy, and indicates the important role of neural components in regulating the pulpal circulation. After administering norepinephrine (0.2 mg/kg/dog), the surface texture of the smooth muscle cells of pulp arterioles reveals marked irregularities, which are correlated with arteriolar contraction. The pericytes in larger postcapillary venules (diameter 20 microm or larger) also show irregularities, whereas no changes are seen in the pericytes of either smaller postcapillary venules or capillaries. The intercellular spaces of pericytes in the postcapillary venules are wide enough for leukocytes to pass through, and the occasional extravasation of leukocytes through venule walls can be seen under electron microscopy. The microvessels of healthy human dental pulp react weakly to selectins, indicating that apparently healthy dental pulp may be weakly inflamed. In rat dental pulp, CGRP-immunoreactive nerves and nerve terminals containing many granular vesicles supply the postcapillary venules more densely than the arterioles, which suggests the involvement of postcapillary venules in neurogenic inflammation in the dental pulp.

Stretch-induced Retinal Vascular Endothelial Growth Factor Expression Is Mediated by Phosphatidylinositol 3-Kinase and Protein Kinase C (PKC)-ζ but Not by Stretch-induced ERK1/2, Akt, Ras, or Classical/Novel PKC Pathways

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.

Evidence for a role of capillary pericytes in vascular growth of the developing ovine corpus luteum.

Because of rapid growth followed by spontaneous regression, the ovarian corpus luteum (CL) is an excellent model to study angiogenesis in vivo. To evaluate the expression of vascular endothelial growth factor (VEGF) protein during luteal development, ovaries were collected from FSH-stimulated ewes throughout the estrous cycle. VEGF was immunolocalized in tissue sections by using an affinity-purified antibody. VEGF protein localized exclusively to the thecal layer of preovulatory follicles, while the granulosa was devoid of staining. Associated with the periovulatory period was intense expression of VEGF by thecal cells at the basement membrane and subsequent invasion of the granulosa layers by these VEGF-positive cells immediately after ovulation. The early CL showed staining for VEGF in thecal-derived compartments, and strong staining for VEGF was also seen in cells within the granulosa-derived parenchymal lobules. Dual immunohistochemical localization of VEGF and smooth muscle cell alpha-actin indicated that the VEGF-positive cells were capillary pericytes or vascular smooth muscle cells. In another experiment, we quantified proliferation of endothelial cells and pericytes throughout luteal development. Pericytes represented a large proportion of the proliferating cells during the early luteal phase and then decreased dramatically. Perivascular cells, therefore, may play a critical role in angiogenesis that occurs during transformation of the follicle into the highly vascular CL of the sheep. As angiogenesis occurs only at the level of capillaries, and pericytes are integral members of these microvessels, regulation of pericytes may provide a novel mechanism for regulating luteal growth and tissue growth in general.

Glucose Instability Is Associated With a High Level of Circulating P-Selectin

We have previously shown that glycemic instability, as measured by the coefficient of variation for fasting plasma glucose (CV-FPG), is an independent predictor of cardiovascular mortality in type 2 diabetes (1,2). The mechanisms, if any, underlying the association between long-term plasma glucose instability and vascular diseases are difficult to explain. In vitro studies with retinal capillary pericytes have shown that rapid glucose fluctuations in the culture medium induced cellular damage and death by apoptosis (3). Moreover, tubulointerstitial cells exposed to intermittent high glucose concentrations underwent changes in cellular growth, collagen synthesis, and cytokine secretion that were more severe than those observed in cells exposed to stable high-glucose concentrations (4). These data extrapolated …

High glucose and advanced glycation end products induce phospholipid hydrolysis and phospholipid enzyme inhibition in bovine retinal pericytes

In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20–50 mM glucose or 2–20 μM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1- 14C]arachidonate and [Me- 3H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1- 14C]arachidonate and [Me- 3H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A 2 (cPLA 2), acyl-CoA:1-acyl- sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A 2 (cPLA 2) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 μM AGEs. Simultaneously, [Me- 3H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.

Regional variations in the apparent diffusion coefficient and the intracellular distribution of water in rat brain during acute focal ischemia.

The apparent diffusion coefficient of water (ADC) rapidly drops in ischemic tissue after cerebral artery occlusion. This acute drop is thought to be caused by the loss of extracellular fluid and the gain of intracellular fluid. To test the latter possibility, changes in ADC and the size of several cellular compartments were assessed in 3 regions of rat brain at the end of 90 minutes of focal cerebral ischemia. One middle cerebral artery was permanently occluded in 8 Sprague-Dawley rats; sham occlusions were performed in 2 other rats. ADC maps were generated 90 minutes later, and the brains were immediately perfusion fixed. Three regions of interest (ROIs) were defined on the basis of ADC range. Various neuronal, astrocytic, and capillary compartments in each ROI were quantified with light and electron microscopy. At the end of 90 minutes of ischemia, mean ADC was normal in the cortex of sham-operated rats and the contralateral cortex of ischemic rats (ROI-a), 25% lower in the ipsilateral frontoparietal cortex (ROI-b), and 45% lower in the ischemic lateral caudoputamen (ROI-c). At this time, the frequency of swollen astrocytic cell bodies and volume of swollen dendrites and astrocytic processes in neuropil were ROI-a<ROI-b<ROI-c. In ROI-b and ROI-c, 40% and 60% of the neurons, respectively, were shrunken; the shrunken neurons were approximately 25% smaller in ROI-c than in ROI-b. In these areas, many capillary endothelial cells, pericytes, and perivascular foot processes were swollen. The initial lowering of ADC during focal ischemia probably is the result of not only the acute loss of extracellular fluid and concomitant swelling of various cellular compartments but also concurrent neuronal shrinkage.

Retinal vascular architecture is maintained in retinal degeneration: corrosion cast and electron microscope study.

To demonstrate the changes of the retinal vascular architecture in the diffusely degenerated thin retina. Three-week-old weanling Wistar Kyoto rats were divided randomly into two groups. One group (n = 20) was fed a vitamin E-deficient solid diet and the other group (n = 20) was fed a solid rat chow diet. Rats were maintained on their respective diets for 14 months and then killed for scanning electron microscopy of vascular corrosion casts, light and electron microscopy and biochemical determinations. The serum level of vitamin E in the E-deficient rats was 1.0 +/- 0.49 microg/ml, while that in the rats fed a normal diet was 13.7 +/- 1.0 microg/ml (Student's t-test, p = 0.0001). In vitamin E-deficient rats, light microscopy showed degenerated retinas only half as thick as normal. Corrosion casts and scanning electron microscopy revealed that the retinal capillaries of the entire retina were decreased in number and scattered with localised narrowing, calibre irregularity and frequent loop formation. In the posterior pole of the retina, some capillaries clustered into small tortuous knots. However, the two-layered architecture of the capillary network in the retina was maintained. The differences in calibre of retinal capillaries between the vitamin E-deficient and normal rats were statistically significant (p < 0.0001). No remarkable abnormal changes were observed in the large retinal vessels other than arterial calibre differences (p < 0.022). No arteriovenous shunts, crossing defects or microaneurysms were seen. Transmission electron microscopy revealed complete disappearance of the photoreceptor outer and inner segments and nuclei. The retinal pigment epithelium contained lipofuscin granules and retinal capillaries with narrow lumens. The capillary endothelial cells were thickened and had scarce cytoplasmic components with vacuoles and irregularly thickened basement membranes. The capillary pericytes had vacuoles. No abnormalities were seen in the control normal rats. These findings indicate that the decrease in retinal capillaries in vitamin E-deficient rats is secondary to retinal degeneration. It was assumed that the morphological changes in the capillary network reflected structural damage to the retinal vascular cells caused by free radicals and lipid peroxides generated by oxidation. However, even in such severe degeneration the retinal vascular architecture, including the main artery and vein and two-layer capillary networks, was maintained. This is may be because of the basic anatomical arrangement of the blood vessels.

Pharmacological inhibition of diabetic retinopathy: aminoguanidine and aspirin.

Effects of aminoguanidine and aspirin on the development of retinopathy have been examined in 5-year studies of diabetic dogs. Either agent was administered daily in doses of 20-25 mg. kg(-1). day(-1). Because severity of hyperglycemia greatly influences development of the retinopathy, special effort was devoted to maintaining comparable glycemia in experimental and control groups. The retinal vasculature was isolated by the trypsin digest method, and retinopathy was assessed by light microscopy. Diabetes for 5 years resulted, as expected, in saccular capillary aneurysms, pericyte ghosts, acellular capillaries, retinal hemorrhages, and other lesions. Administration of aminoguanidine essentially prevented the retinopathy, significantly inhibiting the development of retinal microaneurysms, acellular capillaries, and pericyte ghosts compared with diabetic controls. Aspirin significantly inhibited the development of retinal hemorrhages and acellular capillaries over the 5 years of study, but had less effect on other lesions. Although diabetes resulted in significantly increased levels of advanced glycation end products (AGEs) (namely, pentosidine in tail collagen and aorta, and Hb-AGE), aminoguanidine had no significant influence on these parameters of glycation. Nitration of a retinal protein was significantly increased in diabetes and inhibited by aminoguanidine. The biochemical mechanism by which aminoguanidine has inhibited retinopathy thus is not clear. Aminoguanidine (but not aspirin) inhibited a diabetes-induced defect in ulnar nerve conduction velocity, but neither agent was found to influence kidney structure or albumen excretion.

Amyloid β(1–42) and its β(25–35) fragment induce in vitro phosphatidylcholine hydrolysis in bovine retina capillary pericytes

We describe the inhibitory effect of full-length Aβ(1–42) and Aβ(25–35) fragment of amyloid-β peptide on phosphatidylcholine (PtdCho) metabolism in bovine retina capillary pericytes. Cell cultures were incubated with Aβs for 24 h. Peroxidation indices (malondialdehyde and lactate dehydrogenase release) significantly increased after 20–50 μM Aβ(1–42) or Aβ(25–35) treatment. In addition, [Me- 3H]choline incorporation into PtdCho strongly decreased while either 3H-choline or 14C-arachidonic acid release from prelabeled cells increased, indicating PtdCho hydrolysis. The effect was very likely due to prooxidant action of both Aβ peptides. Reversed-sequence Aβ(35–25) peptide did not depress 3H-choline incorporation nor stimulate PtdCho breakdown. With addition of Aβs at low concentrations (2–20 μM) to pericytes, marked ultrastructural changes, well connected to metabolic alterations, emerged including shrinkage of cell bodies, retraction of processes, disruption of the intracellular actin network. Cells treated with higher concentrations (50–200 μM) displayed characteristics of necrotic cell death. The data suggest that: (a) Aβ(1–42) and Aβ(25–35) peptides may modulate phospholipid turnover in microvessel pericytes; (b) together with endothelial cells, pericytes could be the target of vascular damage during processes involving amyloid accumulation.

Induction Of Endothelin‐1 Expression By Glucose: An Effect Of Protein Kinase C Activation

Enhanced actions or levels of endothelin‐1 (ET‐1), a potent vasoconstrictor, have been associated with decreased blood flow in the retina and peripheral nerves of diabetic animals and may be related to the development of pathologies in these tissues. Hyperglycemia has been postulated to increase ET‐1 secretion in endothelial cells. We have characterized the mechanism by which elevation of glucose is increasing ET‐1 mRNA expression in capillary bovine retinal endothelial cells (BREC) and bovine retinal pericytes (BRPC). Elevation of glucose, but not mannitol, from 5.5 to 25 mmol/l for 3 days increased membranous protein kinase C (PKC) activities and ET‐1 mRNA in parallel levels by 2‐fold in BREC and BRPC. These effects were reversed by decreasing glucose levels to 5.5 mmol/l for an additional 2 days. Glucose‐induced ET‐1 overexpression was inhibited by a general PKC inhibitor, GF109203X, and a mitogen‐activated protein kinase kinase inhibitor, PD98059, but not by wortmannin, a phosphatidylinositol 3‐kinase inhibitor. By immunoblot analysis, PKC‐beta 2 and ‐delta isoforms in BREC were significantly increased relative to other isoforms in the membranous fractions when glucose level was increased. Overexpression of PKC‐beta 1 and ‐delta isoforms but not PKC‐zeta isoform by adenovirus vectors containing the respective cDNA enhanced in parallel PKC activities, proteins, and basal and glucose‐induced ET‐1 mRNA expression by at least 2‐fold. These results showed that enhanced ET‐1 expression induced by hyperglycemia in diabetes is partly due to activation of PKC‐beta and ‐delta isoforms, suggesting that inhibition of these PKC isoforms may prevent early changes in diabetic retinopathy and neuropathy.

The three-dimensional architecture of retinal blood vessels in KK mice, with special reference to the smooth muscle cells and pericytes.

In the present study, the three-dimensional architecture of retinal vasculature was studied in KK mice, by combined use of resin injection, chemical treatment and scanning electron microscopy (SEM). In particular, Mercox/methylmethacrylate resin-injected eye tissues were subjected in sequence to NaClO immersion, ultrasonication and HCl treatment. The present technological innovation made possible SEM visualization of deeper retinal vasculature. In KK mice, the tunica media of stem arterioles and of first and second order branches consisted of a single layer of spindle-shaped smooth muscle cells provided with spine-like cytoplasmic processes. In addition, there occurred small triangular smooth muscle cells provided with slender cytoplasmic processes. The processes, giving off tiny secondary processes, overlapped with each other, thus forming assemblages around branching sites. Such a structure was particularly prominent for those branching sites where parent arterioles gave rise to their branches in a side arm-like pattern. The third (and occasionally fourth) order branches were surrounded by atypical smooth muscle cells, with considerable dimension of endothelial surface remaining uncovered. Capillary pericytes consisted of fusiform cell bodies and slender cytoplasmic processes. Smooth muscle cells of retinal venules differed from those of arterioles. They were stellate in shape, exhibiting several cytoplasmic processes.

Retinal vascular changes in rats with inherited hypercholesterolemia – Corrosion cast demonstration

Purpose. To demonstrate specific hypercholesterolemic changes in the retinal vascular architecture. Methods. Corrosion casts of 12- to 18-month-old rats with inherited hypercholesterolemia (RICO) and of control Wistar Kyoto (WKy) rats were examined with a scanning electron microscope (SEM). The diameters of the retinal arteries, veins and capillaries were measured in photographs with a caliber micrometer. The capillary branches were counted in the micrographs with the use of Adobe Photoshop. The retinal capillaries were examined by transmission electron microscopy (TEM). Results. SEM examination of the vascular casts of 15-month-old RICO rats showed slight tortuosity of large vessels at the posterior pole of the retina. The precapillary arterioles branching from the major artery were longer and straighter than normal. Retinal capillary changes such as caliber irregularity and narrowing in the capillary network were more severe in 18-month-old RICO rats. The most prominent finding was marked straightening of the capillaries in the inner and outer layers of the capillary network, which looked like fine strings. Intercapillary spaces became wider, and finally capillaries looked scattered. The diameter of the retinal capillaries lumen in RICO rats was significantly narrower than that in WKy rats (p < 0.0001). The capillary branches were fewer in 18-month-old RICO rats than that in 18-month-old WKy rats (p < 0.0001). Neither local stenosis or obstruction in the arterioles and venules nor any arteriovenous crossing defect was seen in young and old RICO rats. Transmission electron microscopy of 16-month-old RICO rat retinas revealed that the capillaries in the inner and outer plexiform layers contained scarce cytoplasmic components, vacuoles in endothelial cells and basement membranes of irregular thickness. Capillary pericytes were swollen and irregular in shape, contained vacuolated mitochondria and scarce cytoplasmic components. Conclusions. These findings indicate that the retinal capillary changes are probably related to hypercholesterolemia.

Brain phagocytes may empty tissue debris into capillaries.

The possibility that brain phagocytes may empty remnants of degenerated neurons into capillaries has been studied in frogs. Degeneration of nerve fibers was brought about by transectioning the optic tract, the tectothalamic and tectoisthmic tracts, the postoptic commissure or the radial nerve. To help identification of phagocytozed degenerated neuronal elements, the transected fibers were filled either with horseradish peroxidase (HRP) or cobaltous-lysine complex. The survival times were 3, 4, 7, 27, 47 and 70 days after the application of the markers. The HRP-labeled structures were identified in 60 microm thick sections using diaminobenzidine as chromogen, while cobalt was precipitated in the form of cobaltous sulfide. Small pieces of these sections were further processed for electron microscopy. In each area of the brain and spinal cord investigated, microglial cells and astrocytic processes containing fragments of degenerated neuronal elements could be seen close to capillaries. In some cases a microglial or astrocytic process pierced the capillary basal lamina and seemingly delivered inclusion bodies into the cytoplasm of capillary endothelial cells and pericytes. In the inclusion bodies, which were usually large vesicles, fragments of HRP or cobalt-labeled or unlabeled membranes with a foamy appearance, or condensed myelin lamellae could be observed. These vesicles protruded the luminal membrane of the endothelial cell that was disrupted in some cases suggesting that the content of the inclusion body was discharged into the lumen of the capillary. These results give support to Penfield's hypothesis (1925) that glial cells may empty phagocytozed materials into capillaries.