Eugene G. Levin

Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration

The effects of enhanced HSP27 expression or expression of a nonphosphorylatable form of HSP27 on the migration of bovine arterial endothelial cells was assessed. Expression of the wild‐type protein enhanced migration by twofold compared to control transfectants, whereas expression of the mutant protein retarded migration by 40%. Since homologs of the small heat shock protein inhibit F‐actin polymerization in vitro and may alter basolateral F‐actin content in vivo, it was postulated that the 27 kDa heat shock protein affects microfilament extension essential for cell motility. Expression of the wildtype protein promoted the generation of long cellular extensions, whereas expression of the dominant negative mutant protein resulted in a marked reduction of lamellipodia and generated aberrant microfilament morphology at the wound edge. Immunofluorescence combined with phalloidin staining demonstrated the colocalization of the HSP27 gene products with lamellipodial microfilament structures. These data suggest that the 27 kDa heat shock protein regulates migration by affecting the generation lamellipodia microfilaments.—Piotrowicz, R. S., Hickey, E., Levin, E. G. Heat shock protein 27 kDa expression and phosphorylation regulates endothelial cell migration. FASEB J. 12, 1481–1490 (1998)

Comparative studies of the fibrinolytic activity of cultured vascular cells

Abstract The fibrinolytic components in cultured endothelial cells (ECs) were examined and compared to those in non-endothelial vascular cells (NEVCs) and in normal and virally-transformed fibroblasts. All vascular cells exhibited plasminogen activator (PA) activity. This activity was low in ECs isolated from rabbit aorta and vena cava but high in NEVCs isolated from these same tissues, indicating that the fibrinolytic potential of the vasculature is not restricted to the endothelium. Low PA activity was also characteristic of ECs obtained from human umbilical cord vein. In contrast, both ECs and NEVCs isolated from bovine aorta and vena cava had high PA activity and were even more active in this respect than chick embryo fibroblast transformed by Rous sarcoma virus. Treatment of all cells with 5 × 10−8 M phorbol myristate acetate, a tumor promoting agent, resulted in a variable but significant stimulation of PA activity. The low PA activity associated with some of the EC cultures correlated with the presence of a cellular inhibitor of fibrinolysis. Inhibitor activity was high in rabbit and human ECs and low in rabbit NEVCs and in chick embryo fibroblasts. No inhibitor activity could be detected in cultures of confluent bovine aortic ECs although there was significant inhibitor activity associated with sparsely populated cultures. The specific activity of PA in ECs also fluctuated with cell density, increasing approximately 20-fold as sparsely plated cells grew to confluency. These observations emphasize 1) that tissue associated fibrinolytic activity represents a balance between the activities of PAs and cellular inhibitors, 2) that the relation between PA and inhibitor may change with the growth state of the cells, and 3) that these activities vary with species of origin and thus are not necessarily indicative of any specific cell type.

Lipoproteins inhibit the secretion of tissue plasminogen activator from human endothelial cells.

We studied the effect of lipoprotein(a) [Lp(a)], low-density lipoprotein (LDL), and high-density lipoprotein (HDL) on tissue plasminogen activator (TPA) secretion from human endothelial cells. At 1 mumol/L, Lp(a) inhibited constitutive TPA secretion by 50% and phorbol myristate acetate- and histamine-enhanced TPA secretion by 40%. LDL and HDL also depressed TPA secretion by 45% and 35% (constitutive) and 40% to 60% (stimulated). TPA mRNA levels were also examined and found to change in parallel with antigen secretion. In contrast to TPA, plasminogen activator inhibitor type-1 secretion and mRNA levels were not affected by any of the three lipoproteins. These results suggest that the interaction of lipoproteins with certain cell-surface binding sites may interfere with the proper production and/or secretion of TPA.

Basolateral Membrane-associated 27-kDa Heat Shock Protein and Microfilament Polymerization*

The in vivo activity of the 27-kDa heat shock protein, a barbed-end microfilament capping protein, may be localized to the plasma membrane. To investigate this putative association, bovine endothelial cells expressing the human wild type or a mutant nonphosphorylatable 27-kDa heat shock protein were subjected to subcellular fractionation and immunoblot analysis. The 25-kDa endogenous bovine homolog and both exogenous gene products partitioned with cytosolic or plasma membrane components, indicating that phosphorylation is not required for membrane association. Phorbol ester treatment resulted in phosphorylation of only membrane-associated 25-kDa and wild type 27-kDa heat shock protein and did not induce redistribution. In a second fractionation protocol, streptavidin-agarose precipitation of extracts prepared from cells biotinylated at either the apical or basal surface localized membrane 25- and 27-kDa heat shock protein exclusively to the basolateral surface. Stimulation of transfectants expressing the wild type 27-kDa heat shock protein resulted in its phosphorylation and a doubling in the amount of membrane-associated F-actin precipitated, whereas the mutant protein decreased the amount of F-actin precipitated. These data suggest that membrane-associated 25- and 27-kDa heat shock proteins inhibit the generation of basolateral microfilaments and that phosphorylation releases this inhibition.

THE 27-KDA HEAT SHOCK PROTEIN FACILITATES BASIC FIBROBLAST GROWTH FACTOR RELEASE FROM ENDOTHELIAL CELLS

Basic fibroblast growth factor is an important mitogenic and angiogenic factor that stimulates endothelial cell growth and migration. This hormone is not secreted via the classical vesicular pathway, and the identification of intracellular proteins that facilitate its release remains lacking. Transfection and expression of the 27-kDa human heat shock protein in bovine arterial endothelial cells doubles the rate of estrogen-induced basic fibroblast growth factor secretion, preferentially inducing the release of high molecular weight forms of the hormone. The secreted basic fibroblast growth factor is mitogenic to breast adenocarcinoma cells cultured in the conditioned medium obtained from the transfected endothelial cells. In contrast, decreasing the level of the endogenous heat shock protein homolog with an antisense vector markedly decreases basic fibroblast growth factor release. Anti-heat shock protein or anti-basic fibroblast growth factor antibodies co-precipitate both proteins from endothelial cell extracts, demonstrating a direct association between the two proteins. This interaction is likely to be an important step in the mechanism of basic fibroblast growth factor secretion.

Accelerated growth and senescence of arterial endothelial cells expressing the small molecular weight heat-shock protein HSP27.

Bovine arterial endothelial cells were stably transfected with the human wild‐type (wt) HSP27 or a mutant gene (mu) encoding a nonphosphorylatable form of the protein. At early passage both cultural and cellular morphology were similar, although the vacuole content in wtHSP27 was much higher than muHSP27 cells. As the cultures aged, wtHSP27 cells became large, polymorphic, highly vacuolated, and reached senescence before muHSP27 transfected cultures, which remained small and polygonal with few detectable vacuoles. Vector control cells showed an intermediate phenotype. Tritiated thymidine incorporation studies were performed with multiple wtHSP27 and muHSP27 clones and the results compared with 11 vector control clones. The results showed an average increase in growth rate for the wtHSP27 cells of 3.0 ± 0.6 times. The growth rate of eight muHSP27 clones showed a slight decrease. Estradiol treatment of endothelial cells resulted in an increase in both bovine and human HSP27, with peak expression at 100 nM. Treatment of the vector‐transfected cells with 100 nM estradiol resulted in a 1.44 ± 0.18 fold increase in growth rate, which was blocked by expression of muHSP27. These data demonstrate a role for HSP27 in controlling the growth rate of endothelial cells in an estrogen‐responsive manner.—Piotrowicz, R. S., Weber, L. A., Hickey, E., Levin, E. G. Accelerated growth and senescence of arterial endothelial cells expressing the small molecular weight heat‐shock protein HSP27. FASEBJ. 9, 1079‐1084 (1995)

Serum-mediated suppression of cell-associated plasminogen activator activity in cultured endothelial cells

Abstract Cultured bovine aortic endothelial cells accumulate high levels of plasminogen activator (PA) activity at confluency. This cell-associated PA (PA c ) activity remains high when the cells are maintained without refeeding. Addition of fresh growth medium to these cultures results in a 70–80% decrease in this activity. The decrease is apparent within 30 min, is complete by 2 hr and is initiated by the fetal calf serum (FCS) in the medium. The decrease is transient, with PA c returning to control levels within 4 days, and occurs each time these confluent cultures are fed; it is not associated with the initiation of DNA synthesis. The magnitude of the decrease in PA c activity depends on the concentration of the added serum, with 0.1% FCS causing a 50% decrease. Maximum suppression is observed with 1% FCS. The decrease in PA c activity is not due to serum-mediated stimulation of secretion since PA secretion is also suppressed by fresh serum. Neither changes in the activity of cellular inhibitors nor the presence of contaminating serum inhibitors appears to account for the observed loss of activity. The suppressor activity of serum is nondialyzable but heat-labile. Treatment of serum with diisopropylfluorophosphate (DFP) or hirudin does not reduce its suppressor activity, suggesting that neither thrombin nor other serine proteases are involved. This PA-suppressor activity is not a unique property of FCS since sera from other species, as well as bovine platelet-poor plasma (PPP), are effective in reducing PA c activity. These observations indicate that the fibrinolytic activity of cultured endothelial cells is modified by routine culture manipulations and suggest that production of PA by the endothelium itself may be subject to negative regulation by suppressor molecules in the blood.

Characterization of a plasminogen activator secreted by cultured bovine aortic endothelial cells

A plasminogen activator was purified from the serum-free conditioned medium of bovine aortic endothelial cell cultures by chromatography on zinc chelate-agarose and benzamidine-CH-Sepharose. The final material consisted of a main fibrinolytically active component with Mr 30,000 and a minor component with Mr 41,000. It was obtained with a yield of 60%, a purification factor of 35 and a purity of 25-50%. The activity of this plasminogen activator was completely neutralized by antibodies to human urokinase but not by antibodies against human tissue plasminogen activator. Purified tissue plasminogen activator from bovine heart, however, was completely neutralized by antibodies against human tissue plasminogen activator but unaffected by antibodies to human urokinase. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like. These findings indicate that bovine aortic endothelial cells in culture secrete mainly a urokinase-like plasminogen activator, and not a tissue-type plasminogen activator as was generally assumed.

Dual activities of 22-24 kDA basic fibroblast growth factor: inhibition of migration and stimulation of proliferation.

Basic fibroblast growth factor (FGF2) is synthesized as four isoforms with molecular weights of 24, 22.5, 22, and 18 kDa, with each of the three higher molecular weight forms (hmwFGF2) produced by the initiation of translation at one of three upstream CUG codons. We have shown that bovine arterial endothelial cells export the high molecular weight forms of FGF2 (hmwFGF2) in a 17β‐estradiol–dependent manner (Piotrowicz et al., 1997, J Biol Chem 272:7042–7047). To determine whether the hmwFGF2 forms affected cell behavior after release, we evaluated the effect of recombinant hmwFGF2 on the growth and migration of endothelial cells and mammary carcinoma cells (MCF‐7). Treatment with the recombinant protein resulted in the inhibition of endothelial cell migration by 45% and MCF‐7 cell migration by 70%. HmwFGF2‐dependent inhibition was observed when endothelial cell migration was stimulated by 18 kDa FGF2 or vascular endothelial growth, and MCF cell migration was stimulated with insulin‐like growth factor. In each case, inclusion of an antibody against the 55 amino acid amino terminal end of 24 kDa FGF2 abrogated the inhibition of migration, while antibodies to the 18 kDa FGF2 domain had no effect. When endothelial cells were cultured under conditions which promoted export of hmwFGF2, a 40% decrease in motility was observed which was reversed by the antibodies to the 24 kDa FGF2. Thus, both recombinant and endogenously produced hmwFGF2 are capable of inhibiting migration. In contrast to the ubiquitous effect on migration, hmwFGF2 had no effect on endothelial cell growth but stimulated MCF‐7 growth equally as well as the 18 kDa FGF2 (threefold). Antibodies to the 18 kDa domain of 24 kDa FGF2 blocked the growth‐promoting activity of hmwFGF2, but those to the amino terminal end were ineffective. These data suggest that hmwFGF2 has dual activities, an inhibitory effect on cell migration and a growth‐stimulating effect. The two activities can be localized to different parts of hmwFGF2: inhibitory activity to the amino terminal 55 amino acids (which are absent from the 18 kDa FGF2) and growth‐promoting activity to the 18 kDa domain. Therefore, the ratio of hmwFGF2 and 18 kDa FGF2 in the extracellular space may provide a mechanism of control for angiogenesis and mammary tumor development. J Cell Physiol 178:144–153, 1999.

Properties of plasminogen activators produced by endothelial cells

The plasma fibrinolytic system is responsible for the dissolution of blood clots. It thus promotes continued circulation of the blood through an unobstructed vascular bed. Unfortunately, in spite of more than 30 years of research, the identity and origin of the plasminogen activator (PA) in blood responsible for initiating and controlling this process remains uncertain (1). Much of this confusion results from the presence of multiple, functionally distinct types of PA in blood (1, 2), their inherent instability (3), and the presence of molecules in blood that inhibit them (4). In an attempt to circumvent these problems, we have begun to characterize the PAs produced by cultured endothelial cells (ECs) since available information suggests that the physiologically important vascular PA may originate from the endothelium itself (5). The purpose of this review is to summarize (1) observations that implicate the endothelium in vascular fibrinolysis; (2) experiments that delineate the fibrinolytic components produced by cultured ECs; and (3) studies indicating that the production and activity of these PAs may be influenced by exogenous agents, including some molecules associated with the thrombus itself.