Mary E. Gerritsen

Functional heterogeneity of vascular endothelial cells

This review has highlighted some of the well-described differences in endothelial cells derived from different sites of the vascular tree. In presenting a select group of endothelial properties, there was no intention to imply that these are the only properties of endothelial cells that exhibit heterogeneity. Nonetheless, having described endothelial heterogeneity in regard to a number of defined properties, we are left with persistent questions including: Are these divergent properties of endothelial cells fixed? Alternatively, can we alter the properties of endothelial cells by affecting the signals from the environment? A number of reports strongly suggest that endothelial cells are capable of acquiring new properties. Stewart and Wiley investigated the role of the neural tissue environment on the differentiation of brain capillary endothelial cells. These authors transplanted ectopic sites, i.e. vascular segments of brain from very young quail embryos to chick coeliac cavity, and a quail somites to chick brain ventricles. The distinctive morphology of quail cells provided a cell marker to differentiate host from graft. The results of this study demonstrated that mesenteric or somatic vessels growing into grafted brain developed functional, structural and histochemical features specific for neural capillaries. Conversely, capillaries in mesodermal tissue that had been grafted to the brain were devoid of the neural capillary characteristics, indicating that brain vessels do not form a barrier when they are made to vascularize non-neural tissue. Milici and Carley reported that bovine adrenal capillary cells cultured on plastic exhibited occasional diaphragmed fenestrations and no transendothelial channels. However, if these same cells were cultured on a basement membrane (matrix) laid down by MDCK cells (a canine nephron epithelial cell line), the cells responded by increasing the number of diaphragmed fenestrations and transendothelial channels. This cell culture study supported an earlier whole animal study in which the importance of the epithelium and/or epithelial basal lamina in the maintenance of endothelial ultrastructure was demonstrated in a developmental study of rat intestinal capillaries. In this earlier study, it was noted that epithelial development coincided with the formation of fenestrations by the endothelium. Enzymatic activities of endothelial cells can also be altered by environmental signals. For example, primary cerebral microvascular endothelial cells exhibit barrier features and are enriched in gamma-glutamyl transpeptidase activity, yet rapidly lose the activity when subcultured.(ABSTRACT TRUNCATED AT 400 WORDS)

12(R)-hydroxyeicosatrienoic acid, a potent chemotactic and angiogenic factor produced by the cornea

Human and bovine corneal epithelial cytochrome P450 convert arachidonic acid to compound D [12(R)-hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid], a metabolite with inflammatory properties including vasodilatation and breakdown of the blood-aqueous barrier. Angiogenic properties of the endogenous compound D and the synthetic enantiomers DR and DS were examined using the corneal micropocket technique. The synthetic compound DR was as active as the endogenously formed compound D. Neovascularization of the cornea was found in all the implants containing as little as 0.5 micrograms of compound DR. In contrast, the stereoisomer DS at the same concentration (0.5 micrograms) was inactive. Since angiogenesis can be secondary to a local inflammatory response, we evaluated the effects of compound DR and its stereoisomer DS on human neutrophil chemotaxis by using a modified Boyden chamber technique. DR, but not DS, was found to be a potent chemotactic factor, exhibiting dose-dependent neutrophil chemotaxis with significant responses observed at doses as low as 10(-11) M, a concentration at which leukotriene B4 does not exhibit significant chemotactic activity. Therefore, compound D produced by the cornea may qualify as an intrinsic corneal angiogenic factor which, in association with other inflammatory mechanisms, account for the growth of new vessels in the cornea that appear in chronic inflammation or in the reparative stages of an acute process.

Metabolism of opioid peptides by cerebral microvascular aminopeptidase M

Aminopeptidase M (EC 3.4.11.2), which can degrade low molecular weight opioid peptides, has been reported in both peripheral vasculature and in the CNS. Thus, we have studied the metabolism of opioid peptides by membrane-bound aminopeptidase M derived from cerebral microvessels of hog and rabbit. Both hog and rabbit microvessels were found to contain membrane-bound aminopeptidase M. At neutral pH, microvessels preferentially degraded low molecular weight opioid peptides by hydrolysis of the N-terminal Tyr1-Gly2 bond. Degradation was inhibited by amastatin (I50 = 0.2 microM) and bestatin (10 microM), but not by a number of other peptidase inhibitors including captopril and phosphoramidon. Rates of degradation were highest for the shorter peptides (Met5- and Leu5-enkephalin) whereas beta-endorphin was nearly completely resistant to N-terminal hydrolysis. Km values for the microvascular aminopeptidase also decreased significantly with increasing peptide length (Km = 91.3 +/- 4.9 and 28.9 +/- 3.5 microM for Met5-enkephalin and Met5-enkephalin-Arg6-Phe7, respectively). Peptides known to be present within or in close proximity to cerebral vessels (e.g., neurotensin and substance P) competitively inhibited enkephalin degradation (Ki = 20.4 +/- 2.5 and 7.9 +/- 1.6 microM, respectively). These data suggest that cerebral microvascular aminopeptidase M may play a role in vivo in modulating peptide-mediated local cerebral blood flow, and in preventing circulating enkephalins from crossing the blood-brain barrier.

Flavonoids: inhibitors of cytokine induced gene expression.

Flavonoids demonstrate a remarkable spectrum of biochemical activities which critically focuses on the immune and inflammatory response, including direct inhibitory effects on tyrosine and serine-threonine protein kinases, phospholipases, cyclooxygenases and lipoxygenases (rev. in Middleton Jr. and Kandaswami (1992)). However, our laboratory recently demonstrated that flavonoids can also exert anti-inflammatory effects by inhibiting cytokine induced gene expression (Gerritsen et al., 1995). In this earlier study we reported that apigenin and certain structurally related hydroxyflavones inhibited tumor necrosis factor (TNF), interleukin-1 (IL-1), lipopolysaccharide, and interferon-γ (IFNγ) induced expression of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The effects of apigenin were reversible, occurred in the first 30 minutes of coincubation with the cytokine, and appeared to be at the level of transcription. Additionally, apigenin inhibited the cytokine induced upregulation of several other inflammatory genes including IL-6, IL-8, and cy-clooxygenase-2.

Microvascular Endothelial Cells: Isolation, Identification, and Cultivation

Publisher Summary This chapter discusses isolation, identification, and cultivation of microvascular endothelial cells. The development of successful techniques for the isolation, identification, and culture of large-vessel endothelium has paved the way for the extension of these methods to microvascular endothelial cells. In isolation and culture of retinal microvessel endothelial cells, bovine eyes are obtained from a local slaughterhouse and transported to the laboratory on ice. The culture of heart microvessel endothelial cells has provided an important tool as to the role of the vascular endothelium of cardiac muscle in a number of physiologic and pathophysiologic functions, including prostaglandin synthesis. One of the major difficulties in the isolation and culture of microvascular endothelial cells is the identification of the putative endothelial cells.

Glucocorticoid-mediated alterations in fluidity of rabbit cardiac muscle microvessel endothelial cell membranes: influences on eicosanoid release

Experiments were conducted to determine effects of the synthetic glucocorticoid, dexamethasone, on the lipid fluidity of cultured rabbit cardiac muscle microvessel endothelial cells and the possible role(s) for altered fluidity in the steroid inhibition of cellular eicosanoid production. Following a sixteen hour exposure to 10(-7) M dexamethasone, membranes prepared from treated cells exhibited a decreased fluidity compared to their control counterparts, as assessed by steady-state fluorescence polarization techniques using 1,6-diphenyl-1,3,5-hexatriene (DPH). Examination of the effects of temperature on the anisotropy values of DPH using Arrhenius plots revealed consistent differences in the steroid treated cells over the entire temperature range (40-5 degrees C). These dexamethasone-dependent fluidity changes were associated with increases in the cholesterol/phospholipid ratio of membrane lipids. Restoration of membrane fluidity to control values with the fluidizing agent, 2-(2-methoxyethoxy)ethyl-8-(cis- 2-n-octylcyclopropyl)octanoate (A2C), partially reversed dexamethasone induced inhibition of A23187-stimulated eicosanoid release. These observations suggest that at least part of dexamethasones inhibitory actions on eicosanoid generation in microvessel endothelial cells are mediated by alterations in membrane composition and fluidity.

Insulin binding and effects of insulin on glucose uptake and metabolism in cultured rabbit coronary microvessel endothelium.

Abstract Microvascular endothelial cells were isolated from rabbit cardiac tissue, and cultivated by standard tissue culture techniques. The conversion of [U-14C]glucose to CO2 and lipids was significantly enhanced by insulin treatment. Insulin stimulated uptake of 2-[3H]deoxyglucose and enhanced the transport of 3-O[3H]methyl-D-glucose. Specific binding of [125I]insulin to RCME cells, displaceable by cold insulin, was also observed. These data demonstrate that insulin is capable of regulating metabolic activities in coronary microvascular endothelium.

Prostaglandin synthesis and release from cultured human trabecular-meshwork cells and scleral fibroblasts

Human trabecular-meshwork (HTM) cells in culture convert arachidonic acid to two products: PGE2 and 6-keto PGF1 alpha. Prostaglandin PGE2 was the major product of arachidonic-acid metabolism. The synthesis and release of PGE2 and 6-keto PGF1 alpha was inhibited by a 15-min pre-treatment with indomethacin (5 X 10(-6) M) or a 4-24-hr incubation with 10(-7) M dexamethasone. The effects of dexamethasone could be prevented by cycloheximide (0.5 micrograms ml-1), or actinomycin D (2 micrograms ml-1). Prostaglandin E2 synthesis and release by these HTM cells from two different individuals could be stimulated by bradykinin, arachidonic acid and the calcium ionophore, A23187.

Regulation of hexose transport in cultured bovine retinal microvessel endothelium by insulin

In microvessel endothelium obtained from young calf retinas, insulin stimulated the uptake of 2-deoxy-D-glucose in a dose-dependent manner. The maximal level of glucose uptake (50-100% over basal values) was observed at 1 ng ml-1 insulin. The effects of insulin on glucose uptake were time-dependent, with optimum stimulation of glucose transport observed after 90 min of insulin treatment. The effects of insulin were prevented by 15 min pretreatment with 2 micrograms ml-1 cycloheximide or by 2 micrograms ml-1 actinomycin D, suggesting a role for new protein synthesis in the actions of insulin on glucose transport in this cell type. These results demonstrate that cultured retinal microvessel endothelial cells exhibit an insulin-sensitive glucose transport system.

Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells

In the present study we determined the uptake and disposition of glucose in serum-deprived rabbit coronary microvessel endothelial (RCME) cells. RCME cells exhibited stereospecific hexose uptake inhibited by cytochalasin B. Pretreatment of the RCME cells with potassium cyanide or 2,4-dinitrophenol inhibited 2-deoxyglucose uptake but not 3-O-methylglucose transport. A major proportion (30-60%) of the 2-deoxyglucose present in the RCME cells was not phosphorylated. These two observations suggested that the rate-limiting step in the uptake of 2-deoxyglucose was not transport but rather the phosphorylation of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. When glucose-deprived cells were incubated 2 hr with [U-14C]glucose the disposition of the label was as follows: glycogen 60%, acid-soluble fraction 30%, and lipid less than 5%. In contrast glucose-fed cells exhibited lower overall glucose incorporation, and a slightly different disposition: glycogen 45%, acid-soluble fraction 50%, and lipid 5%. Glucose-deprived RCME cells also exhibited greater basal levels of 2-deoxyglucose uptake compared to glucose-fed cells. RCME cells incubated in the absence of glucose and serum for 16 hr exhibited dose-dependent insulin stimulation of hexose uptake and subsequent metabolism to macromolecules (i.e., glycogen and the acid-soluble fraction). Significant effects of insulin were observed with concentrations as low as 2 x 10(-10) M, well within the physiological range. In contrast, cells preincubated in serum-free culture medium containing 5.5 mM glucose did not exhibit insulin-enhanced hexose uptake or glucose metabolism (even at doses as high as 10(-7) M). These studies indicate that the effects of insulin on rabbit coronary microvascular endothelial cell glucose uptake and metabolism require both serum and glucose deprivation.