Role of Nitric Oxide in Megakaryocyte Function.

Megakaryocytes are the main members of the hematopoietic system responsible for regulating vascular homeostasis through their progeny platelets, which are generally known for maintaining hemostasis. Megakaryocytes are characterized as large polyploid cells that reside in the bone marrow but may also circulate in the vasculature. They are generated directly or through a multi-lineage commitment step from the most primitive progenitor or Hematopoietic Stem Cells (HSCs) in a process called "megakaryopoiesis". Immature megakaryocytes enter a complicated development process defined as "thrombopoiesis" that ultimately results in the release of extended protrusions called proplatelets into bone marrow sinusoidal or lung microvessels. One of the main mediators that play an important modulatory role in hematopoiesis and hemostasis is nitric oxide (NO), a free radical gas produced by three isoforms of nitric oxide synthase within the mammalian cells. In this review, we summarize the effect of NO and its signaling on megakaryopoiesis and thrombopoiesis under both physiological and pathophysiological conditions.

Platelets stimulate programmed death-ligand 1 expression by cancer cells: Inhibition by anti-platelet drugs.

BACKGROUND: Platelets facilitate hematogenous metastasis in part by promoting cancer cell immunoevasion, although our understanding of platelet function in modulating the adaptive immune system in cancer is limited. A major negative regulator of the adaptive response is the immune checkpoint protein Programmed Death Ligand 1 (PD-L1). OBJECTIVES: As platelets secrete factors that may increase PD-L1 expression, we investigated whether they up-regulate cancer cell PD-L1, thus promoting immunoevasion, and whether common anti-platelet drugs inhibit this process. METHODS: Platelets were isolated from human volunteers. A549 lung, PD-L1 null A549, and 786-O renal cancer cells were incubated with and without platelets, and cancer cell PD-L1 expression was measured by qPCR and flow cytometry. Additionally, platelet-cancer cell incubations were performed in the presence of common anti-platelet drugs, and with growth factor neutralizing antibodies. Following incubation with platelets, A549 were co-cultured with T-cells and interleukin-2 (IL-2) levels were measured by flow cytometry as a marker of T-cell activation. RESULTS: Platelets increased PD-L1 mRNA and surface protein expression by A549 and 786-0 cells. Combined neutralization of VEGF and PDGF prevented the platelet-induced up-regulation of PD-L1 by A549, as did the anti-platelet drug eptifibatide. A549 incubated with platelets demonstrated a reduced ability to activate human T-cells, an effect reversed by eptifibatide. CONCLUSIONS: As platelets promote immunoevasion of the adaptive immune response by increasing cancer cell PD-L1 expression and as anti-platelet drugs prevent this immunoevasive response, the investigation of anti-platelet drugs as adjuvant therapy to immune checkpoint inhibitors may be warranted in the treatment of cancer.

Preferential interaction of platelets with prostate cancer cells with stem cell markers.

BACKGROUND: Prostate cancer (PCa) may be initiated by CD133+/CD44+ expressing stem cell-like cells (PCSC), which are also thought to drive metastasis. Platelets also contribute to metastasis via tumor cell-induced platelet aggregation (TCIPA), which in part enhances cancer cell invasion. Moreover, activated platelets secrete stromal derived growth factor-1α (SDF-1α) that can mobilize CSCs via the CXCR4 receptor. However, the potential reciprocal interactions between CSCs and platelets have not been investigated. OBJECTIVE: To characterize the mechanisms behind PCSC-platelet interaction. METHODS: Fluorescence Activated Cell Sorting was utilized to separate DU145 and PC3 PCa cells into CD133+/CD44+, CD133+/CD44-, CD44+/CD133-, and CD133-/CD44- subpopulations and to measure their CXCR4 surface expression. PCa subpopulation TCIPA experiments were performed using aggregometry and immunoblot was used to measure prothrombin. Platelet SDF-1α secretion was measured by ELISA. Modified-Boyden chamber assays were used to assess the role of SDF-1α:CXCR4 pathway in platelet-PCSC interactions. RESULTS: DU145 and PC3 expressing both CD133 and CD44 stem cell markers accounted for only small fractions of total cells (DU145: CD133+/CD44+ 3.44 ± 1.45% vs. CD133+/CD44- 1.56 ± 0.45% vs. CD44+/CD133- 68.19 ± 6.25% vs. CD133-/CD44- 20.36 ± 4.51%). However, CD133+ subpopulations induced the greatest amount of aggregation compared to CD44+/CD133- and double-negative DU145, and this aggregation potency of CD133+ PCa cells corresponded with high levels of prothrombin expression. Additionally, CD133+ subpopulations expressed significantly higher level of CXCR4 compared to CD133-/CD44- and CD44+/CD133-. Disruption of SDF-1α:CXCR4 pathway reduced platelet-induced PCSC invasion. CONCLUSIONS: CD133+/CD44+ and CD133+/CD44- PCSCs have highest platelet aggregation potency, which could be attributed to their increased prothrombin expression. Reciprocally, platelet-derived SDF-1α stimulates PCSC invasion.

The role of the androgen receptor in prostate cancer-induced platelet aggregation and platelet-induced invasion.

BACKGROUND: Metastatic prostate cancer progresses from a hormone sensitive androgen receptor expressing phenotype to a hormone insensitive androgen receptor-independent subtype with low overall survival. Human platelets contribute to metastasis via tumor cell-induced platelet aggregation, which in part enhances cancer cell invasion. Given the more aggressive nature of hormone insensitive prostate cancer, we hypothesized that androgen receptor-negative prostate cancer cells exhibit higher platelet aggregation potency and invasive response compared to cells with androgen receptor. OBJECTIVE: To characterize the role of androgen receptors in prostate cancer-induced platelet aggregation and platelet-induced invasion. METHODS: Tumor cell-induced platelet aggregation experiments were performed with platelets from healthy human donors and benign prostate (RWPE-1) and prostate cancer cell lines positive (LNCaP) and negative for androgen receptor (DU145 and PC3). Immunoblot measured prostate cancer prothrombin. Modified Boyden chamber invasion assays and zymography were performed to assess the effects of platelets on prostate cancer cell invasion and matrix metalloproteinase (MMP) expression, respectively. RESULTS: Androgen receptor-positive prostate cancer cell lines failed to induce platelet aggregation. However, androgen receptor-inhibited and -negative cell lines all induced platelet aggregation, which was abolished by dabigatran. Androgen receptor-inhibited and -negative cell lines demonstrated greater expression of prothrombin than androgen receptor-positive cells. Platelets enhanced invasion and MMP-2 and -9 expression by androgen receptor-inhibited and negative prostate cancer cells, but not that of the androgen receptor-positive cells. CONCLUSIONS: Androgen receptor loss within prostate cancer results in increased thrombogenicity due to upregulation of prothrombin expression. Reciprocally, platelets enhance invasion of androgen receptor-negative prostate cancer cells via increased MMP expression.

STAT3 but Not HIF-1α Is Important in Mediating Hypoxia-Induced Chemoresistance in MDA-MB-231, a Triple Negative Breast Cancer Cell Line.

Hypoxia-induced chemoresistance (HICR) is a well-recognized phenomenon, and in many experimental models, hypoxia inducible factor-1α (HIF-1α) is believed to be a key player. We aimed to better understand the mechanism underlying HICR in a triple negative breast cancer cell line, MDA-MB-231, with a focus on the role of HIF-1α. In this context, the effect of hypoxia on the sensitivity of MDA-MB-231 cells to cisplatin and their stem-like features was evaluated and the role of HIF-1α in both phenomena was assessed. Our results showed that hypoxia significantly increased MDA-MB-231 resistance to cisplatin. Correlating with this, intracellular uptake of cisplatin was significantly reduced under hypoxia. Furthermore, the stem-like features of MDA-MB-231 cells increased as evidenced by the significant increases in the expression of ATP-binding cassette (ABC) drug transporters, the proportion of CD44⁺/CD24- cells, clonogenic survival and cisplatin chemoresistance. Under hypoxia, both the protein level and DNA binding of HIF-1α was dramatically increased. Surprisingly, siRNA knockdown of HIF-1α did not result in an appreciable change to HICR. Instead, signal transducer and activator of transcription 3 (STAT3) activation was found to be important. STAT3 activation may confer HICR by upregulating ABC transporters, particularly ABCC2 and ABCC6. This study has demonstrated that, in MDA-MB-231 cells, STAT3 rather than HIF-1α is important in mediating HICR to cisplatin.

Functional assessment of von Willebrand factor expression by cancer cells of non-endothelial origin.

Von Willebrand factor (VWF) is a highly adhesive procoagulant molecule that mediates platelet adhesion to endothelial and subendothelial surfaces. Normally it is expressed exclusively in endothelial cells (ECs) and megakaryocytes. However, a few studies have reported VWF detection in cancer cells of non-endothelial origin, including osteosarcoma. A role for VWF in cancer metastasis has long been postulated but evidence supporting both pro- and anti-metastatic roles for VWF has been presented. We hypothesized that the role of VWF in cancer metastasis is influenced by its cellular origin and that cancer cell acquisition of VWF expression may contribute to enhanced metastatic potential. We demonstrated de novo expression of VWF in glioma as well as osteosarcoma cells. Endothelial monolayer adhesion, transmigration and extravasation capacities of VWF expressing cancer cells were shown to be enhanced compared to non-VWF expressing cells, and were significantly reduced as a result of VWF knock down. VWF expressing cancer cells were also detected in patient tumor samples of varying histologies. Analyses of the mechanism of transcriptional activation of the VWF in cancer cells demonstrated a pattern of trans-activating factor binding and epigenetic modifications consistent overall with that observed in ECs. These results demonstrate that cancer cells of non-endothelial origin can acquire de novo expression of VWF, which can enhance processes, including endothelial and platelet adhesion and extravasation, that contribute to cancer metastasis.

CYP1B1 inhibition attenuates doxorubicin-induced cardiotoxicity through a mid-chain HETEs-dependent mechanism.

Doxorubicin (DOX) has been reported to be a very potent and effective anticancer agent. However, clinical treatment with DOX has been greatly limited due to its cardiotoxicity. Furthermore, several studies have suggested a role for cytochrome P450 1B1 (CYP1B1) and mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in DOX-induced cardiac toxicity. Therefore, we hypothesized that DOX induced cardiotoxicity is mediated through the induction of CYP1B1 and its associated mid-chain HETEs metabolite. To test our hypothesis, Sprague-Dawley rats and RL-14 cells were treated with DOX in the presence and absence of 2,3',4,5'-tetramethoxystilbene (TMS), a selective CYP1B1 inhibitor. Thereafter, cardiotoxicity parameters were determined using echocardiography, histopathology, and gene expression. Further, the level of mid-chain HETEs was quantified using liquid chromatography-electron spray ionization-mass spectrometry. Our results showed that DOX induced cardiotoxicity in vivo and in vitro as evidenced by deleterious changes in echocardiography, histopathology, and hypertrophic markers. Importantly, the TMS significantly reversed these changes. Moreover, the DOX-induced cardiotoxicity was associated with a proportional increase in the formation of cardiac mid-chain HETEs both in vivo and in our cell culture model. Interestingly, the inhibition of cardiotoxicity by TMS was associated with a dramatic decrease in the formation of cardiac mid-chain HETEs suggesting a mid-chain HETEs-dependent mechanism. Mechanistically, the protective effect of TMS against DOX-induced cardiotoxicity was mediated through the inhibition of mitogen activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB). In conclusion, our study provides the first evidence that the inhibition of CYP1B1 and mid-chain HETE formation attenuate DOX-induced cardiotoxicity.

The role of platelets in the tumor microenvironment: From solid tumors to leukemia.

Platelets are increasingly being recognized for promoting tumor growth and metastasis. Many cells derived from solid tumors have the ability to aggregate platelets, and this ability correlates with their metastatic potential. Over the past half century, our understanding of tumor cell-induced platelet aggregation (TCIPA) has grown beyond the simple concept that tumor cell-containing microthrombi mechanically embolize the microvasculature. Tumor cell-activated platelets secrete a multitude of factors that reciprocally act on tumor cells, as well as other cells within the tumor microenvironment; thus, affecting both parenychma and tumor-associated stroma. In this review, we summarize the current knowledge of tumor cell-platelet interactions and their influence on the tumor microenvironment, including how these interactions impact neoplastic epithelial cells, endothelial cells, pericytes, fibroblasts, immune cells, and early metastatic niches. In addition, we review the current knowledge of platelet-cancer cell interactions within hematological malignancies and speculate on how platelets may influence the leukemic microenvironment. This article is part of a Special Issue entitled: Tumor Microenvironment Regulation of Cancer Cell Survival, Metastasis, Inflammation, and Immune Surveillance edited by Peter Ruvolo and Gregg L. Semenza.

Proteomic profile of aminoglutethimide-induced apoptosis in HL-60 cells: Role of myeloperoxidase and arylamine free radicals.

In this study, the cellular effects resulting from the metabolism of aminoglutethimide by myeloperoxidase were investigated. Human promyelocytic leukemia (HL-60) cells were treated with aminoglutethimide (AG), an arylamine drug that has a risk of adverse drug reactions, including drug-induced agranulocytosis. HL-60 cells contain abundant amounts of myeloperoxidase (MPO), a hemoprotein, which catalyzes one-electron oxidation of arylamines using H2O2 as a cofactor. Previous studies have shown that arylamine metabolism by MPO results in protein radical formation. The purpose of this study was to determine if pathways associated with a toxic response could be determined from conditions that produced protein radicals. Conditions for AG-induced protein radical formation (with minimal cytotoxicity) were optimized, and these conditions were used to carry out proteomic studies. We identified 43 proteins that were changed significantly upon AG treatment among which 18 were up-regulated and 25 were down-regulated. The quantitative proteomic data showed that AG peroxidative metabolism led to the down-regulation of critical anti-apoptotic proteins responsible for inhibiting the release of pro-apoptotic factors from the mitochondria as well as cytoskeletal proteins such as nuclear lamina. This overall pro-apoptotic response was confirmed with flow cytometry which demonstrated apoptosis to be the main mode of cell death, and this was attenuated by MPO inhibition. This response correlated with the intensity of AG-induced protein radical formation in HL-60 cells, which may play a role in cell death signaling mechanisms.

Occlusive lung arterial lesions in endothelial-targeted, fas-induced apoptosis transgenic mice.

Pulmonary arterial hypertension (PAH) is a lethal disease that is characterized by functional and structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance and ultimately right heart failure. In experimental models of pulmonary hypertension, endothelial cell (EC) apoptosis is a necessary trigger for the development of obliterative lung arteriopathy, inducing the emergence of hyperproliferative and apoptosis-resistant vascular cells. However, it has not been established whether EC apoptosis is sufficient for the induction of complex lung arteriolar lesions. We generated a conditional transgenic system in mice to test the hypothesis that lung endothelial cell apoptosis is sufficient to induce a PAH phenotype. The Fas-induced apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter (i.e., EFIA mice), and administration of a small molecule dimerizing agent, AP20187, resulted in modest pulmonary hypertension, which was associated with obliterative vascular lesions localized to distal lung arterioles in a proportion of transgenic mice. These lesions were characterized by proliferating cells, predominantly CD68 macrophages. Although endothelial cell apoptosis was also seen in the kidney, evidence of subsequent arteriopathy was seen only in the lung. This model provides direct evidence that lung endothelial cell apoptosis acts as a trigger to initiate a PAH phenotype and provides initial insight into the potential mechanisms that underlie a lung-specific arterial response to endothelial injury.

Pharmacological regulation of platelet factors that influence tumor angiogenesis.

In addition to maintaining hemostasis, platelets play an important pathological role driving tumor growth and metastasis. One mechanism by which platelets contribute to tumor growth and metastasis is their potent promotion of angiogenesis. This is accomplished in large part by the numerous factors stored, generated, and released by platelets that have the potential to influence every stage of angiogenesis. In this review, we provide an overview of the many platelet-secreted pro- and anti-angiogenic factors. We examine the basic science and clinical evidence supporting their contributions to tumor angiogenesis. Finally, we review the pharmacological regulation of their release from platelets and discuss the potential of anti-platelet drugs as adjuvant anti-angiogenesis therapy.

Temporal and pharmacological characterization of angiostatin release and generation by human platelets: implications for endothelial cell migration.

Platelets play an important role in thrombosis and in neo-vascularisation as they release and produce factors that both promote and suppress angiogenesis. Amongst these factors is the angiogenesis inhibitor angiostatin, which is released during thrombus formation. The impact of anti-thrombotic agents and the kinetics of platelet angiostatin release are unknown. Hence, our objectives were to characterize platelet angiostatin release temporally and pharmacologically and to determine how angiostatin release influences endothelial cell migration, an early stage of angiogenesis. We hypothesized anti-platelet agents would suppress angiostatin release but not generation by platelets. Human platelets were aggregated and temporal angiostatin release was compared to vascular endothelial growth factor (VEGF). Immuno-gold electron microscopy and immunofluorescence microscopy identified α-granules as storage organelles of platelet angiostatin. Acetylsalicylic acid, MRS2395, GPIIb/IIIa blocking peptide, and aprotinin were used to characterize platelet angiostatin release and generation. An endothelial cell migration assay was performed under hypoxic conditions to determine the effects of pharmacological platelet and angiostatin inhibition. Compared to VEGF, angiostatin generation and release from α-granules occurred later temporally during platelet aggregation. Consequently, collagen-activated platelet releasates stimulated endothelial cell migration more potently than maximally-aggregated platelets. Platelet inhibitors prostacyclin, S-nitroso-glutathione, acetylsalicylic acid, and GPIIb/IIIa blocking peptide, but not a P2Y12 inhibitor, suppressed angiostatin release but not generation. Suppression of angiostatin generation in the presence of acetylsalicylic acid enhanced platelet-stimulated endothelial migration. Hence, the temporal and pharmacological modulation of platelet angiostatin release may have significant consequences for neo-vascularization following thrombus formation.

Pharmacologic protein kinase Cα inhibition uncouples human platelet-stimulated angiogenesis from collagen-induced aggregation.

Platelets promote angiogenesis by releasing angiogenesis-regulating factors from their α-granules upon aggregation. This effect has both physiologic and pathologic significance as it may contribute to carcinogenesis. Platelet α-granule release and aggregation are regulated, in part, via protein kinase C (PKC) α and ß signaling. Our study investigated the effects of PKC inhibition on aggregation, angiogenesis-regulator secretion from α-granules, and platelet-stimulated angiogenesis. We hypothesized that selective PKCα inhibition may preferentially suppress angiogenesis-regulator secretion from α-granules but not aggregation, limiting platelet-stimulated angiogenesis. Human platelets were aggregated in the presence of conventional PKC inhibitors myr-FARKGALRQ and Ro 32-0432 (2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyridol[1,2-α]indol-3-yl}-3-(1-methyl-1H-indol-3-yl)maleimide). Immunofluorescence microscopy of PKC translocation was used to determine the specificity of PKC-inhibitor targeting. Enzyme-linked immunosorbent assay was used to measure vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1) release from platelets. Platelet effects on angiogenesis were tested using a capillary-formation assay. Ro 32-0432, but not the peptide inhibitor myr-FARKGALRQ (myristoylated-pseudosubstrate peptide inhibitor), inhibited aggregation in a concentration-dependent manner, while both Ro 32-0432 and myr-FARKGALRQ preferentially suppressed VEGF over TSP-1 secretion. Suppression of angiogenesis-regulator release occurred at inhibitor concentrations that did not significantly affect aggregation. Immunofluorescence microscopy revealed that PKCα targeting to α-granules is inhibited when angiogenesis-regulator secretion is uncoupled from aggregation. At concentrations that uncoupled α-granule release from aggregation, Ro 32-0432 and myr-FARKGALRQ inhibited platelet-stimulated angiogenesis. Hence, selective PKCα inhibition suppresses angiogenesis-regulator release from platelet α-granules with minimal effects on aggregation. Thus, selective PKCα inhibitors may have pharmacologic significance to regulate platelet-promoted angiogenesis.

Angiostatin inhibits endothelial MMP-2 and MMP-14 expression: a hypoxia specific mechanism of action.

Angiostatin is an angiogenesis inhibitor in part generated by and released from platelets. Since platelets upon thrombus formation can give rise to areas of hypoxia, we investigated the effects of angiostatin on endothelial cell migration and apoptosis during hypoxia. Human microvascular endothelial cells (HMVEC-L) were exposed to angiostatin under normoxic or hypoxic conditions. Apoptosis was measured by flow-cytometry. HMVEC-L migration was studied using a modified Boyden Chamber assay, in which migration is MMP-dependent. MMP-2, MMP-14, and VEGF levels were measured using immunoblot, Q-PCR and ELISA. During hypoxia HMVEC-L were protected from angiostatin-induced apoptosis due to increased hypoxia-induced VEGF expression. However, MMP-dependent migration of HMVEC-L was inhibited by angiostatin under hypoxic but not normoxic conditions. Angiostatin decreased MMP-2 at the gene and protein levels only in HMVEC-L exposed to hypoxia. A similar result was obtained for MMP-14. Higher angiostatin concentrations, as would be seen during thrombosis, induced HMVEC-L apoptosis, which was not rescued by VEGF. Under hypoxic conditions angiostatin's primary anti-angiogenic mechanism is likely inhibition of endothelial cell MMP-dependent endothelial cell migration. Only at higher concentrations does angiostatin induce endothelial cell death. This study identifies a novel angiostatin anti-angiogenesis mechanism that is only triggered under pathological-like conditions.

Platelet-associated angiogenesis regulating factors: a pharmacological perspective.

Platelets, in addition to maintaining hemostasis, also stimulate angiogenesis by generating and releasing, upon activation, factors that promote the growth of new blood vessels. To date, at least 20 angiogenesis-regulating factors have been identified in platelets, including both promoters and inhibitors. Platelet-derived angiogenesis regulators promote angiogenesis during wound healing, tumor growth, and in response to ischemia. Within platelets, angiogenesis regulators are primarily stored in α-granules, but are also found in the cytosol or derived from membrane lipids. Their release can be inhibited pharmacologically by anti-platelet agents, which consequently suppress platelet-stimulated angiogenesis. Several years ago, our research group discovered that platelets generate the angiogenesis inhibitor angiostatin independent of the activation state of platelets, and that platelet-derived angiostatin serves to limit the angiogenesis-stimulating effects of platelets. In this review, we summarize the current knowledge of platelet-associated angiogenesis regulators, how they impact angiogenesis, and how they are controlled pharmacologically.

VEGF masks BNIP3-mediated apoptosis of hypoxic endothelial cells.

Hypoxia results in the apoptotic death of myocytes, neurons, and epithelial cells, through the actions of Bcl-2 and Nineteen kilodalton Interacting Protein-3 (BNIP3). On the contrary, endothelial cells are especially adept at surviving conditions of oxygen deprivation via up-regulation of vascular endothelial growth factor (VEGF) the most potent endothelial survival factor. Both VEGF and BNIP3 expression are transcriptionally regulated by hypoxia inducible factor and may antagonize each other's affects in endothelial cells (ECs). Since factors that promote and inhibit apoptosis may be expressed at the same time in endothelial cells under hypoxic conditions, we decided to investigate whether VEGF and BNIP3 have opposing actions in endothelial cells. Human microvascular endothelial cells were exposed to hypoxic conditions in a Billups-Rothenburg chamber. Under hypoxic conditions BNIP3 expression by endothelial cells increased as measured by real-time PCR and immunoblot. After 48 h of hypoxia, EC apoptosis was assessed by flow cytometry and was lower than in corresponding normoxia serum starved controls. The increase in EC survival under hypoxic conditions corresponded with an increase in the expression of VEGF. Under normoxic conditions adenoviral BNIP3 over-expression promoted apoptosis of ECs; however, recombinant VEGF (100 pg/ml) antagonized the BNIP3 apoptosis promoting affects. SiRNA knockdown of VEGF expression by hypoxic ECs resulted in increased apoptosis with a concomitant increase in BNIP3 expression. SiRNA knockdown of BNIP3 expression by hypoxic ECs reduced the increase in EC apoptosis as a result of VEGF knockdown. We conclude that under hypoxic conditions VEGF counteracts and masks the apoptosis promoting affects of BNIP3.

Role of apoptosis in pulmonary hypertension: from experimental models to clinical trials.

Pulmonary arterial hypertension (PAH) is a progressive and lethal disease that has a strong female predominance, often affecting the young. Current therapies are mostly vasodilator agents, and while mainly addressing the endothelial dysfunction that has been widely reported in this disease, they may be less effective in treating arterial remodeling. The lung pathology of PAH is characterized by medial hypertrophy and intimal hyperplasia of muscular arteries as well as plexiform lesions, that lead to a widespread narrowing or obliteration of the pulmonary arteriolar bed. However, the pathogenesis of the functional and structural abnormalities of the lung microcirculation in PAH is poorly understood. Perhaps the greatest advancement in the last decade has been the discovery of the "PAH gene," bone morphogenetic receptor 2 (Bmpr2), however how the loss-of-function mutations in Bmpr2 lead to PAH is unclear. The BMPR2 pathway has recently been shown to mediate survival signaling in endothelial cells (EC), and thus the reduced activity will favor endothelial apoptosis, likely increasing the susceptibility to endothelial injury in response to various environmental triggers. EC apoptosis has been implicated as an initiating event in experimental PAH, leading either directly to the degeneration of pre-capillary arterioles or to the selection of hyperproliferative, apoptosis-resistant ECs that may contribute to "angioproliferative" plexiform lesions. The idea that EC apoptosis may play a central role in the initiation and progression of PAH suggests that therapeutic strategies aimed at endothelial repair and regeneration of ECs may be uniquely effective in the treatment of this disease. Preclinical evaluation and validation on the use of endothelial progenitor cells (EPCs) for the prevention and reversal of experimental PAH is reviewed and the design of a "first in man" clinical trial to assess the safety and efficacy of a combined EPC and endothelial NO-synthase gene therapy for patients that are refractory to standard therapies is discussed.

Ceramide promotes apoptosis in chronic myelogenous leukemia-derived K562 cells by a mechanism involving caspase-8 and JNK.

Ceramide is a sphingolipid that activates stress kinases such as p38 and c-JUN N-Terminal Kinase (JNK). Though Chronic Myelogenous Leukemia (CML) derived K562 cells resist killing by short chain C2-ceramide, we report here that longer chain C6-ceramide promotes apoptosis in these cells. C6-ceramide induces cleavage of Caspase-8 and Caspase-9, but only Caspase-8 is required for apoptosis. The sphingolipid killed CML derived KBM5 cells and, to a lesser extent, imatinib-resistant KBM5-STI cells suggesting that BCR-ABL can not completely block C6-ceramide-induced apoptosis but the kinase may regulate the process. BCR-ABL is known to suppress Protein Phosphatase 2A (PP2A) in CML cells. While C6-ceramide can activate PP2A in acute leukemia cells, the sphingolipid did not activate the phosphatase in K562 cells. C6-ceramide did not activate p38 kinase but did promote JNK activation and phosphorylation of JUN. Inhibition of JNK by pharmacological agent protected K562 cells from C6-ceramide suggesting that JNK plays an essential role in C6-ceramide mediated apoptosis. Furthermore, the sphingolipid promoted MCL-1 phosphorylation by a mechanism that, at least in part, involves JNK. The findings presented here suggest that Caspase-8, JNK, and perhaps MCL-1 may play important roles in regulating cell death and may represent new targets for therapeutic strategies for CML.

Role of metalloproteinases in platelet function.

Platelets contain and release matrix metalloproteinases (MMPs), their inhibitors (TIMPs) and disintegrin metalloproteinases (ADAMs) including MMP-1, MMP-2, MMP-3, MMP-9, MT1-MMP (MMP-14), ADAM-10, ADAM-17, ADAMTS-13, TIMP-1, TIMP-2 and TIMP-4. These proteins exert several effects regulating platelet functions such as agonist-stimulated platelet adhesion and aggregation, tumour cell-induced platelet aggregation and platelet-leukocyte aggregation. In this review, mechanisms of MMPs, TIMPs and ADAMs on platelets are discussed.

Protein kinase C delta mediates platelet-induced breast cancer cell invasion.

Platelets play an important role in carcinogenesis, but the underlying molecular mechanisms remain poorly understood. To investigate the effects of platelets on in vitro invasion of MCF7 human breast cancer cells, human MCF7 cells were used to study their interactions with platelets using aggregometry and cell invasion chambers. Zymography and quantitative polymerase chain reaction (PCR) were used to study matrix metalloproteinases (MMPs), whereas Western blot was used to study protein kinase C (PKC) delta in MCF7 cells. We observed that platelets promoted invasion of MCF7 cells (3-fold increase, p<0.05, n=3) and that this process correlated with a dramatic increase in MMP-9 (8 fold-increase, p<0.001, n=3), which is known to facilitate cancer cell invasion. Because both platelets and MCF7 cells have been shown to release MMP-9, we investigated the cellular source that accounted for this increase. The time course and the use of specific protein synthesis inhibitors demonstrated that most of the increase in MMP-9 levels derived from de novo synthesis of this protease by cancer cells. Furthermore, platelets activated PKCdelta in MCF7 cells after 1 h of incubation (18.45+/-4.75% increase, p<0.05, n=4-7), which, in turn, led to an up-regulation of MMP-9 mRNA (from 60+/-20 to 1040+/-100 pg, p<0.001, n=3) and protein levels (18-fold increase, p<0.001, n=3), with the subsequent cell invasion-promoting effects. PKCdelta plays a crucial role in transducing the invasion-promoting effects of platelets in breast cancer cells, and the specific inhibition of PKCdelta may be a strategy to decrease platelet-mediated cancer cell invasion.