Darrell H. Carney

Phosphoinositides in mitogenesis: Neomycin inhibits thrombin-stimulated phosphoinositide turnover and initiation of cell proliferation

Thrombin stimulates 32Pi incorporation into phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bis-phosphate (PIP2), and phosphatidylinositol (PI), and initiates DNA synthesis in hamster (NIL) fibroblasts at a half-maximal concentration of 125 ng/ml. Neomycin, which binds PIP2 and PIP, inhibits both thrombin-stimulated initiation of cell proliferation and 32P pI incorporation into at concentrations above 2 mM without affecting thrombin binding, thymidine uptake, or cellular protein synthesis. At lower concentrations, neomycin inhibits thrombin-stimulated release of inositol 1,4,5-trisphosphate (IP3), by selectively binding PIP2, but does not inhibit 32P incorporation into PI or initiation of DNA synthesis. Phosphoinositide recycling and diacylglycerol release therefore appear necessary for initiation of cell proliferation by thrombin. IP3-stimulated Ca++ mobilization may not be required for thrombin mitogenesis, however, since neomycin can block IP3 release without inhibiting initiation.

Evidence that microtubule depolymerization early in the cell cycle is sufficient to initiate DNA synthesis

Microtubule disrupting drugs initiated DNA synthesis in serum-free cultures of nonproliferating fibroblast-like cells. The addition of colchicine to chick, mouse and human fibroblasts in serum-free medium stimulated thymidine incorporation at least twofold, with a half-maximal concentration of 1 X 10(-7) M. This stimulation represented up to 75% of the maximal stimulation by thrombin and was paralleled by an increase in the percentage of labeled nuclei. Other microtubule disrupting drugs showed similar stimulation, whereas lumicolchicine had no effect. Indirect immunofluorescent staining of tubulin showed a correlation between microtubule depolymerization and initiation of DNA synthesis by these drugs. A 2 hr treatment with 10(-6) M colchicine caused complete disruption of the microtubular network and stimulated thymidine incorporation (measured 28 hr later) to an even greater extent than continuous colchicine exposure. A similar 2 hr exposure to 10(-6) M colcemid also stimulated thymidine incorporation and led to a 50% increase in cell number. Taxol, a drug which stabilizes cytoplasmic microtubules, blocks initiation of DNA synthesis by colchicine, indicating that microtubule depolymerization is necessary for this initiation. To determine if microtubule depolymerization is involved in stimulation of DNA synthesis by other growth factors, highly purified human thrombin was added to cells with or without colchicine. In no case did colchicine plus thrombin increase DNA synthesis above that of the maximal stimulation by thrombin alone. Furthermore, pretreatment of cultures with taxol (5 micrograms/ml) inhibited approximately 30% of the stimulation of thymidine incorporation by thrombin. Together, these studies demonstrate that microtubule depolymerization is sufficient to initiate both DNA synthesis and events leading to cell division and suggest that microtubule depolymerization may be a required step in initiation of cell proliferation by growth factors such as highly purified human thrombin.

Microtubule stabilization by taxol inhibits initiation of DNA synthesis by thrombin and by epidermal growth factor

Several observations have suggested that microtubule depolymerization might act as a regulator of cell proliferation. To determine whether microtubule depolymerization is required for growth-factor-induced initiation of DNA synthesis, we treated serum-free cultures of mouse embryo cells with taxol to stabilize their microtubules and measured the initiation of DNA synthesis by thrombin and epidermal growth factor (EGF). Pretreatment of quiescent cultures of mouse embryo cells with 10 microgram/ml taxol inhibited up to 60% of the thrombin-stimulated and 47% of the EGF-stimulated DNA synthesis. This inhibition was dose-dependent for taxol concentrations from 0.3 to 20 microgram/ml. Control experiments showed that taxol did not simply affect uptake of nucleotides, glucose or amino acids, nor did it nonspecifically affect protein synthesis, cell morphology or cell viability. Taxol did not affect binding and internalization of 125I-thrombin or 125I-EGF indicating that the drug does not alter receptor number, affinity or distribution after growth factor binding. Taxol also did not affect the proteolytic activity of thrombin. Thus it appears that the inhibitory effects of taxol are mediated by a direct effect of taxol on microtubules. To determine at what point microtubule stabilization was interrupting the initiation signal, we added taxol at various times after addition of thrombin or EGF. Taxol addition during the first 8 hr after growth factor addition inhibited initiation, but after 8 hr had little if any effect. These results confirm that taxol was not nonspecifically affecting transport or metabolism required for DNA synthesis and indicate that thrombin and EGF may initiate cell proliferation through a gradual microtubule depolymerization or rearrangement that is necessary to commit cells to a replicative cycle.

In vivo degradation of porous poly(propylene fumarate)/poly(DL-lactic-co-glycolic acid) composite scaffolds

This study investigated the in vivo degradation of poly(propylene fumarate) (PPF)/poly(DL-lactic-co-glycolic acid) (PLGA) composite scaffolds designed for controlled release of osteogenic factors. PPF/PLGA composites were implanted into 15.0mm segmental defects in the rabbit radius, harvested after 12 and 18 weeks, and analyzed using histological techniques to assess the extent of polymer degradation as well as the tissue response within the pores of the scaffolds. Polymer degradation was limited to micro-fragmentation of the scaffold at the ends and edges of the implant at both 12 and 18 weeks. The tissue within the pores of the scaffold consisted of fibrous tissue, blood vessels and some inflammatory cells. In areas where polymer breakdown was evident, an increased inflammatory response was observed. In contrast, areas of bone ingrowth into the polymer scaffold were characterized by minimal inflammatory response and polymer degradation. Our results show that minimal degradation of porous PPF occurs within 18 weeks of implantation in a rabbit model. Further, the in vivo degradation data of porous PPF/PLGA scaffolds are comparable with earlier obtained in vitro data.

Controlled release of an osteogenic peptide from injectable biodegradable polymeric composites.

Poly(D,L-lactic-co-glycolic acid)/poly(ethylene glycol) (PLGA/PEG) blend microparticles loaded with the osteogenic peptide TP508 were added to a mixture of poly(propylene fumarate) (PPF), poly(propylene fumarate)-diacrylate (PPF-DA), and sodium chloride (NaCl) for the fabrication of PPF composite scaffolds that could allow for tissue ingrowth as well as for the controlled release of TP508 when implanted in an orthopedic defect site. In this study, PPF composites were fabricated and the in vitro release kinetics of TP508 were determined. TP508 loading within the PLGA/PEG microparticles, PEG content within the PLGA/PEG microparticles, the microparticle content of the PPF composite polymer component, and the leachable porogen initial mass percent of the PPF composites were varied according to a fractional factorial design and the effect of each variable on the release kinetics was determined for up to 28 days. Each composite formulation released TP508 with a unique release profile. The initial release (release through day 1) of the PLGA/PEG microparticles was reduced upon inclusion in the PPF composite formulations. Day 1 normalized cumulative mass release from PPF composites ranged from 0.14+/-0.01 to 0.41+/-0.01, whereas the release from PLGA/PEG microparticles ranged from 0.31+/-0.02 to 0.58+/-0.01. After 28 days, PPF composites released 53+/-4% to 86+/-2% of the entrapped peptide resulting in cumulative mass releases ranging from 0.14+/-0.01 microg TP508/mm(3) scaffold to 2.46+/-0.05 microg TP508/mm(3) scaffold. The results presented here demonstrate that PPF composites can be used for the controlled release of TP508 and that alterations in the composites composition can lead to modulation of the TP508 release kinetics. These composites can be used to explore the effects varied release kinetics and dosages on the formation of bone in vivo.

Organ-derived microvessel endothelial cells exhibit differential responsiveness to thrombin and other growth factors☆

To investigate the relationship between endothelial cells and organ-associated vascular physiology, microvascular endothelial cells were isolated from murine brain, lung, and liver tissues. During culture, these endothelial cells maintained certain differentiated characteristics common to all endothelial cells, but also showed organ-specific characteristics, with distinct patterns of responsiveness to various growth factors. Microvascular endothelial cells from all organs responded to endothelial cell growth factor (ECGF), but lung (LE-1) and brain (MBE-12) endothelial cells showed different responsiveness to thrombin (10-60 nM), combinations of thrombin and ECGF, or thrombin and extracellular matrix. Liver sinusoidal endothelial cells (HSE) were relatively unresponsive to thrombin, but were the most responsive of the endothelial cells to EGF. Endothelial cells isolated from lung and brain, where fluxes in vascular permeability are observed following injury, showed dramatic morphological alterations in response to nanomolar concentrations of thrombin. These cells also exhibited the highest amount of 125I-thrombin binding at these concentrations. Scatchard analysis of 125I-thrombin binding indicated that LE cells have the highest affinity for thrombin, followed by MBE, with HSE exhibiting significantly lower affinity. The binding of 125I-thrombin to these cells was inhibited by the TR-9 monoclonal antibody directed against fibroblast high-affinity thrombin receptors involved in thrombin-stimulated mitogenesis. The results suggest that the differences in growth stimulation observed between organ-derived endothelial cells in response to thrombin, ECGF, and EGF may relate to differential expression of receptors for these factors. These observations demonstrate yet another aspect of the functional heterogeneity of the microvascular endothelium.

THROMBIN RECEPTOR EXPRESSION AND RESPONSIVENESS OF HUMAN MONOCYTIC CELLS TO THROMBIN IS LINKED TO INTERFERON-INDUCED CELLULAR DIFFERENTIATION

Human thrombin has been shown to stimulate monocyte chemotaxis, phagocytosis, and interleukin (IL8) production, but the mechanisms responsible for stimulation are not well defined. In some cells, thrombin stimulation of proliferation appears to require both cleavage of the proteolytically activated receptor for thrombin (PAR1) and activation of a nonproteolytically activated thrombin receptor (N‐PAR), while in others activation of either receptor alone may be sufficient for stimulation. We, therefore, have initiated studies to address thrombin receptor expression and cell responsiveness to thrombin in interferon gamma (IFNγ)‐differentiated and nondifferentiated U937 monocytic cells. Northern blot analysis shows that PAR1 expression is upregulated upon differentiation. Experiments with biotinylated and 125I‐thrombin show that specific thrombin binding is dramatically increased by differentiation although it is not clear if this binding is to PAR1 or to a separate binding component such as N‐PAR which is present on fibroblasts and other cells. Addition of thrombin at concentrations of 1–10 μg/ml (30–300 nM, concentrations where specific thrombin binding is observed) stimulates proliferation of IFNγ‐differentiated U937 cells but not of undifferentiated U937 cells. Thrombin also stimulates interleukin‐6 (IL6) production in IFNγ‐differentiated U937 cells. Moreover, thrombin induces high levels of IL6, interleukin‐1β (IL1β), and tumor necrosis factor‐α (TNFα) production by peripheral blood mononuclear cells (PBMC) and monocytes. These results show that differentiated U937 cells and mature PBMC are responsive to thrombin whereas nondifferentiated U937 are not. Further, this responsiveness appears to correlate with expression of PAR1 and to a dramatic increase in specific thrombin binding. That thrombin stimulates cytokine production and proliferation in populations of differentiated monocytes suggests that thrombin may be an important regulator of inflammation and wound healing. J. Cell. Physiol. 177:76–84, 1998.

Chronic hypoxia attenuates VEGF signaling and angiogenic responses by downregulation of KDR in human endothelial cells.

Coronary artery disease results in progressive vascular stenosis associated with chronic myocardial ischemia. Vascular endothelial growth factor (VEGF) stimulates endothelial cell angiogenic responses to revascularize ischemic tissues; however, the effect of chronic hypoxia on the responsiveness of endothelial cells to VEGF remains unclear. We, therefore, investigated whether hypoxia alters VEGF-stimulated signaling and angiogenic responses in primary human coronary artery endothelial (HCAE) cells. Exposure of HCAE cells to hypoxia (1% O(2)) for 24 h decreased VEGF-stimulated endothelial cell migration ( approximately 82%), proliferation ( approximately 30%), and tube formation. Hypoxia attenuated VEGF-stimulated activation of endothelial nitric oxide (NO) synthase (eNOS) ( approximately 72%) and reduced NO production in VEGF-stimulated cells from 237 +/- 38.8 to 61.3 +/- 28.4 nmol/l. Moreover, hypoxia also decreased the ratio of phosphorylated eNOS to total eNOS in VEGF-stimulated cells by approximately 50%. This effect was not observed in thrombin-stimulated cells, suggesting that hypoxia specifically inhibited VEGF signaling upstream of eNOS phosphorylation. VEGF-induced activation of Akt, ERK1/2, p38, p70S6 kinases, and S6 ribosomal protein was also attenuated in hypoxic cells. Moreover, VEGF-stimulated phosphorylation of VEGF receptor-2 (KDR) at Y996 and Y1175 was decreased by hypoxia. This decrease correlated with a 70 +/- 12% decrease in KDR protein expression. Analysis of mRNA from these cells showed that hypoxia reduced steady-state levels of KDR mRNA by 52 +/- 16% and decreased mRNA stability relative to normoxic cells. Our findings demonstrate that chronic hypoxia attenuates VEGF-stimulated signaling in HCAE cells by specific downregulation of KDR expression. These data provide a novel explanation for the impaired angiogenic responses to VEGF in endothelial cells exposed to chronic hypoxia.

Thrombin peptide, TP508, stimulates angiogenic responses in animal models of dermal wound healing, in chick chorioallantoic membranes, and in cultured human aortic and microvascular endothelial cells

The alpha-thrombin peptide, TP508, accelerates the healing of full-thickness wounds in both normal and ischemic skin. In wounds treated with TP508, a pattern of increased vascularization is consistently observed both grossly and microscopically when compared to wounds treated with saline. One possible mechanism by which the peptide accelerates wound healing is by promoting revascularization of granulation tissue at the injured site. To evaluate the angiogenic potential of TP508, the peptide was tested in the chick embryo chorioallantoic membrane (CAM), where it increased the density and size of CAM blood vessels relative to controls. Additionally, TP508 stimulated chemokinesis and chemotaxis in a dose-dependent fashion in cultured human aortic and human microvascular endothelial cells. Taken together, these in vivo and in vitro data support an angiogenic role for TP508 in wound healing. A working model is presented to explain how this 23-amino-acid peptide, which lacks proteolytic activity, is generated during wound healing and contributes to the nonproteolytic functions associated with alpha-thrombin during tissue repair.

Thrombin regulates the expression of proangiogenic cytokines via proteolytic activation of protease-activated receptor-1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types, including monocytes and endothelial cells, involved in the control of angiogenesis. Different cytokines produced by mononuclear cells have been implicated in angiogenic processes associated with tissue repair and certain human malignancies. We have previously shown that thrombin enhances proliferative responses in T lymphocytes. More recently, we reported that interferon-gamma-differentiated monocytes have increased expression of protease-activated receptor-1 (PAR-1) and increased thrombin binding. Since cytokines may be involved directly and indirectly in angiogenesis, we initiated studies to determine thrombin effects on the induction of cytokines, such as interleukin (IL)-1 and IL-6, in human mononuclear cells. IL-1 and IL-6 protein expression was significantly enhanced by thrombin (P<.05), as determined by enzyme-linked immunosorbent assay (ELISA). Treating mononuclear cells with the PAR-1 peptide, SFLLRN, has effects similar to those of thrombin. Thus, it appears that these thrombin effects are mediated through activation of PAR-1. These results confirm that thrombin is a strong activator of monocytes and could be involved in angiogenesis by inducing cytokines that could enhance the angiogenic process in tissue repair.