James L. Kinsella

Paclitaxel Stent Coating Inhibits Neointimal Hyperplasia at 4 Weeks in a Porcine Model of Coronary Restenosis

BackgroundDespite limiting elastic recoil and late vascular remodeling after angioplasty, coronary stents remain vulnerable to restenosis, caused primarily by neointimal hyperplasia. Paclitaxel, a microtubule-stabilizing drug, has been shown to inhibit vascular smooth muscle cell migration and proliferation contributing to neointimal hyperplasia. We tested whether paclitaxel-coated coronary stents are effective at preventing neointimal proliferation in a porcine model of restenosis. Methods and ResultsPalmaz-Schatz stents were dip-coated with paclitaxel (0, 0.2, 15, or 187 &mgr;g/stent) by immersion in ethanolic paclitaxel and evaporation of the solvent. Stents were deployed with mild oversizing in the left anterior descending coronary artery (LAD) of 41 minipigs. The treatment effect was assessed 4 weeks after stent implantation. The angiographic late loss index (mean luminal diameter) decreased with increasing paclitaxel dose (P <0.0028 by ANOVA), declining by 84.3% (from 0.352 to 0.055, P <0.05) at the highest level tested (187 &mgr;g/stent versus control). Accompanying this change, the neointimal area decreased (by 39.5%, high-dose versus control;P <0.05) with increasing dose (P <0.040 by ANOVA), whereas the luminal area increased (by 90.4%, high-dose versus control;P <0.05) with escalating dose (P <0.0004 by ANOVA). Inflammatory cells were seen infrequently, and there were no cases of aneurysm or thrombosis. ConclusionsPaclitaxel-coated coronary stents produced a significant dose-dependent inhibition of neointimal hyperplasia and luminal encroachment in the pig LAD 28 days after implantation; later effects require further study. These results demonstrate the potential therapeutic benefit of paclitaxel-coated coronary stents in the prevention and treatment of human coronary restenosis.

Protein kinase C regulates endothelial cell tube formation on basement membrane matrix, matrigel

Human umbilical vein endothelial cells differentiate within 12 h to form capillary-like networks of tube structures when the cells are plated on Matrigel, a mixture of basement membrane proteins. Nothing is known about the intracellular signaling events involved in this differentiation. As a first step to define the process, we investigated the possible role of protein kinase C activation by beta-phorbol 12-myristate 13-acetate (PMA) in regulating the formation of the tube structures. In this model, PMA increased tube formation several-fold in a dose-dependent manner with half-maximum stimulation of tube formation at approximately 5 nM PMA. In the absence of serum, essentially little or no tubes were formed on Matrigel unless PMA was added to the medium. Only active phorbol analogs increased tube formation, while the protein kinase C inhibitor, H-7, blocked tube formation. The protein kinase C activators and inhibitors were effective only when added at or just after plating of the cells and did not affect already formed tubes. This study suggests that protein kinase C is involved in the early events of in vitro endothelial cell tube formation on Matrigel.

The Role of Basement Membrane in Angiogenesis and Tumor Growth

Expansion of the tumor-cell mass is dependent on both the degree of tumor vascularization and the rate of angiogenesis. Blood vessel growth is controlled, in part, by the matrix surrounding it, in particular, the basement membrane underlying the endothelium. Here we illustrate that laminin, a major component of basement membrane, has several biologically active sites that can bind to endothelial and tumor cells, and have the ability to regulate angiogenesis and tumor growth. We show that synthetic peptides at two sites in the laminin B1 chain (the RGD and YIGSR sequences) inhibit angiogenesis, whereas a third site in the A chain, designated SIK-VAV, stimulates vessel and tumor cell growth. By developing strategies that promote or inhibit the activities of these sites in laminin, we may obtain methods to inhibit angiogenesis and subsequent tumor growth.

A Slow pH-dependent Conformational Transition Underlies a Novel Mode of Activation of the Epithelial Na+/H+ Exchanger-3 Isoform

Allosteric control of Na+/H+ exchange by intracellular protons ensures rapid and accurate regulation of the intracellular pH. Although this allosteric effect was heretofore thought to occur almost instantaneously, we report here the occurrence of a slower secondary activation of the epithelial Na+/H+ exchanger (NHE)-3 isoform. This slow activation mode developed over the course of minutes and was unique to NHE3 and the closely related isoform NHE5, but was not observed in NHE1 or NHE2. Activation of NHE3 was not due to increased density of exchangers at the cell surface, nor was it accompanied by detectable changes in phosphorylation. The association of NHE3 with the cytoskeleton, assessed by its retention in the detergent-insoluble fraction, was similarly unaffected by acidification. In contrast to the slow progressive activation elicited by acidification, deactivation occurred very rapidly upon restoration of the physiological pH. We propose that NHE3 undergoes a slow pH-dependent transition from a less active to a more active state, likely by changing its conformation or state of association.

Na + /H + exchanger: proton modifier site regulation of activity

The Na+/H+ exchangers (NHE1-6) are integral plasma membrane proteins that catalyze the exchange of extracellular Na+ for intracellular H+. In addition to Na+ and H+ transport sites, NHE has an intracellular allosteric H+ modifier site that increases exchange activity when occupied by H+. NHE activity is also subject to control by a variety of extrinsic factors including hormones, growth factors, cytokines, and pharmacological agents. Many of these factors, working through second messenger pathways acting directly or indirectly on NHE, regulate NHE activity by shifting the apparent affinity of the H+ modifier site to more alkaline or more acid pH. The underlying molecular mechanisms involved in the activation of NHE by the H+ modifier site are poorly understood at this time, but likely involve slow protein conformational changes within a NHE oligomer. In this paper, we present initial experiments measuring intracellular pH-dependent transition rates between active and inactive oligomeric conformations and describe how these transition rates may be important for overall regulation of NHE activity.

Kinetic studies on the stimulation of Na+-H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats.

SummaryNa+−H+ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na+], treatment altered the initial rate of Na+ uptake by increasingVm (hyperthyroid, 18.9±1.1 nmol Na+ · mg−1 · 2 sec−1; normal, 8.9±0.3 nmol Na+ · mg−1 · 2 sec−1), and not the apparent affinityKNa+ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H+] and at a subsaturating [Na+] (1mm), hyperthyroidism resulted in the proportional increase in Na+ uptake at every intravesicular pH measured. A positive cooperative effect on Na+ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H+]0.5 (hyperthyroid, 0.39±0.08 μm; normal, 0.44±0.07 μm) but increased the apparentVm (hyperthyroid, 1.68±0.14 nmol Na+ · mg−1 · 2 sec−1; normal 0.96±0.10 nmol Na+ · mg−1 · 2 sec−1). The uptake of Na+ in exchange for H+ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H+ translocation or debinding was rate limiting for Na+−H+ exchange since Na+−Na+ exchange activity was greater than Na+−H+ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na+−Na+ and Na+−H+ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na+−Na+ and Na+−H+ exchange activity.

Minoxidil inhibits proliferation and migration of cultured vascular smooth muscle cells and neointimal formation after balloon catheter injury

We observed that heterozygous knockout (+/-, KO) of either endothelin-A- (ET(A)) or -B- (ET(B)) receptors significantly reduced the pressor responses to systemically administered endothelin-1 (ET-1) in ET(A) or ET(B) (+/-) KO mice when compared to wild-type (WT) mice (data not shown). Also, we observed that basal mean arterial pressure (MAP) is significantly higher in ET(B) (+/-) (92.7 +/- 1.2 mmHg) (n = 53, p < 0.05) but not ET(A) (+/-) KO mice (70.6 +/- 1.8 mmHg) (n = 23) when compared to their anaesthetized WT littermates (70.1 +/- 0.7 mmHg) (n = 118). A 90 min treatment with either BQ-123 (10 mg/kg), an ET(A)-selective antagonist, or BQ-928 (10 mg/kg), a mixed ET(A)/ET(B) antagonist, administered intraperitoneally, significantly reduced basal MAP of ET(B) (+/-) KO mice almost to the level of their WT treated counterparts (94.9 +/- 4.9 mmHg) (n = 6) vs (+ BQ-123: 59.7 +/- 0.3 mmHg, n = 8); (+ BQ-928: 72.4 +/- 2.6 mmHg, n = 5). It is worthy of note that BQ-123 significantly reduced basal MAP in WT mice but to a lesser extent than in ET(B) (+/-) KO mice (69.6 +/- 2.3 mmHg, n = 8) vs (+ BQ-123: 57.3 +/- 1.4 mmHg, n = 8). In contrast, the ET(B)-selective antagonist, BQ-788 (10 mg/kg i.p.), had no significant effect on MAP even after 90 min of treatment (ET(B) (+/-) KO: (92.3 +/- 2.3 mmHg, n = 6) vs (+ BQ-788: 89.7 +/- 3.1 mmHg, n = 6); WT: (70.5 +/- 3.7 mmHg, n = 7) vs (+ BQ-788: 71.2 +/- 2.0 mmHg, n = 6). Therefore heterozygous KO of either ET(A)- or ET(B)-receptors significantly alters the phenotypic pressor properties of ET-1. We also suggest that there is less ET clearance in ET(B) (+/-) KO mice than in WT mice, which can explain the ET(A)-dependent hypertensive state of the former strain.We evaluated the role of endothelin-B- (ET(B)) receptor-mediated action in the development and maintenance of deoxycorticosterone acetate (DOCA)-salt-induced hypertension, cardiovascular hypertrophy and renal damage, using the spotting lethal (sl) rat which carries a naturally occurring deletion in the ET(B)-receptor gene. Homozygous (sl/sl) rats exhibit abnormal development of the neural crest-derived epidermal melanocytes and the enteric nervous system (ENS), and do not live beyond 1 month because of intestinal aganglionosis and resulting intestinal obstruction. Therefore, the dopamine-beta-hydroxylase (D betaH) promoter was used to direct ET(B) transgene expression in sl/sl rats to support normal ENS development. D betaH-ET(B) sl/sl rats live into adulthood and are healthy, expressing ET(B)-receptor in adrenals and other adrenergic neurons. When homozygous (sl/sl) and wild-type (WT) (+/+) rats, all of which were transgenic, were treated with DOCA and salt for 4 weeks, the homozygous rats exhibited significantly earlier and higher increases in systolic blood pressure than WT rats. The daily oral administration of ABT-627, a selective ET(A)-receptor antagonist, almost completely suppressed the DOCA-salt-induced hypertension in both groups. Renal dysfunction and histological damage induced by DOCA-salt treatment were more severe in homozygous than in WT rats. Increased and marked vascular hypertrophy of the aorta was also observed in homozygous rats, compared with WT rats. Renal and vascular injuries induced by DOCA and salt were significantly improved by ABT-627 administration. We propose that ET(B)-receptor-mediated actions are protective factors in the pathogenesis of DOCA-salt-induced hypertension. ET(A)-mediated actions are at least partly responsible for the increased susceptibility to DOCA-salt-induced hypertension and related tissue injuries in ET(B)-receptor-deficient rats.

Specific Laminin Domains Mediate Endothelial Cell Adhesion, Alignment and Angiogenesis

The stability and integrity of blood vessels is maintained by many factors in the blood and by an important extracellular layer, the basement membrane, which underlies the endothelium of the vessels. Basement membranes are composed of an organized network of collagen (type IV), heparan sulfate proteoglycan, and glycoproteins such as entactin, fibronectin, and laminin. Laminin is one of the most important and abundant substances in basement membranes. It has a direct role in cell attachment, migration, and induction of the differentiated phenotype of many cells. We have examined and defined the role(s) of laminin and its specific cell-binding sites at the biochemical level using an in vitro angiogenic model. This model involves the differentiation of cultured endothelial cells on a laminin rich reconstituted basement membrane matrix, Matrigel, into capillary-like structures. Synthetic peptides derived from sequences in the laminin A and B1 chains (CTFALRGDNP and CDPGYIGSR) were able to block cell attachment to Matrigel and cell-cell alignment (early events in vessel formation), respectively, and thus inhibit subsequent tube formation. The third biologically active site CSRARKQAASIKVAVSADR) induced the endothelial cells to become migratory and invade into the Matrigel, forming sprouts from the primary capillary-like network. This site also induces angiogenesis in the chick chorioallantoic membrane (CAM) assay. Thus, endothelial cells interact with at least three different sites in laminin and these interactions are important in vessel maintenance and repair.

Chapter 13 Hormonal Regulation of Renal Na+-H+ Exchange Activity

Publisher Summary The kidney proximal tubule that carries out transepithelial transport of solutes and fluid, is characterized by cells with polarity. The brush border membrane of the mammalian renal proximal tubule contains the Na + –H + exchanger. The carrier couples Na + flux down its concentration gradient (lumen to cell) to the secretion of H + against its concentration gradient (cell to lumen). This chapter describes some properties of the Na + –H + exchanger in brush border membrane vesicles isolated from the rat renal cortex. It presents some initial experiments, describing how two hormones, thyroid hormones and glucocorticoids, regulate Na + –H + exchange and how exchange activity responds in metabolic acidosis. In addition, the chapter discusses a concept of hormonal hierarchy in the control of renal Na + –H + exchange activity. Na + –H + exchange participates in multiple proximal tubular functions. Na + –H + exchange activity was determined by two independent methods: (1) uptake of 22 Na + energized by H + gradient and the flux of H + into the vesicle energized by an imposed Na + gradient. The chapter enlists the extrinsic factors currently reported to regulate renal Na + –H + exchange activity. The hormones of distinct class and presumed action regulate the activity of the renal proximal tubule Na + –H + exchanger. Multiendocrine inputs and the temporal-dependent hierarchy of hormonal control of Na + –H + exchange represents an initial attempt to develop a concept for the mechanism that regulates the Na + –H + exchange activity.

Gene Expression and Endothelial Cell Differentiation

Regulation of the vascular wall is an essential process that allows normal blood flow and facilitates the exchange of soluble gases, ions and vital macromolecules. Normally all vessels are composed of a nonthrombogenic layer of endothelial cells which line the intimai surface of the vessel walls. The differentiation state of this cell layer is maintained by components (factors) present in the blood the extravascular stroma and the extracellular matrix. The contribution of the matrix to vascular wall homeostasis has been unclear in the past, even though matrix comprise a significant portion of the vasculature. In fact, the endothelium is adherent to a thin, specialized extracellular layer know as a basement membrane. The basement membrane provides not only support and an adhesive surface for the endothelium but also maintains the normal differentiated phenotype of the cell layer. Vessel walls also are comprised of other vascular cells such a smooth muscle cells, pericytes and fibroblasts. The former two also have their own basement membrane, and the latter is surrounded by a collagenous insterstitium (the adventitia) and in some cases elastic fibers. Studies which examine the cells comprising the vessel walls must also evaluate the role of the matrix in the maintenance of its structure as well.