Astrid Parenti

Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

The bradykinin/B1 receptor promotes angiogenesis by up- regulation of endogenous FGF-2 in endothelium via the nitric oxide synthase pathway

Bradykinin (BK) mediates inflammation and contributes to angiogenesis. We assessed the mechanisms for BK contribution to angiogenesis. Nanomolar concentrations of BK induced angiogenesis in rabbit corneas in absence of inflammation. The effect was dose‐dependent and mediated by the B1 receptor. B2 receptor stimulation failed to directly promote vascular growth unless inflammation was induced. Anti‐fibroblast growth factor‐2 (FGF‐2) antibody blocked the effect of BK or B1 receptor agonist. In postcapillary venular endothelial cells (CVEC), B2 receptor activation induced inositol phosphate turnover and calcium transients, whereas the B1 receptor was coupled to nitric oxide synthase (NOS) up‐regulation and activation and cGMP increase. Differential RT‐PCR and Western blot analysis revealed FGF‐ 2 up‐regulation in cells exposed to BK or to the selective B1 agonist, whereas the B2 agonist was without effect. Consistently, BK and the B1 but not the B2 agonist exerted a proliferative effect on CVEC, which was prevented by anti‐FGF‐2 antibody and by NOS inhibition. These results demonstrate that BK is angiogenic despite its proinflammatory activity and that the B1 receptor is involved. The B1 receptor is coupled to NOS activation and FGF‐2 up‐regulation, events not shared by the B2 receptor activation.

The heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and modulates gelatinase and TIMP-2 production in endothelial cells

The hypothesis that thrombospondin‐1 (TSP‐1) can exert opposite effects on angiogenesis depending on the functional status of its domains/fragments was investigated. In the rabbit cornea, TSP‐1 inhibited angiogenesis induced by fibroblast growth factor‐2 (FGF‐2). However, when tested per se, TSP‐1 was able to elicit an angiogenic response comparable to that induced by FGF‐2. Induction of angiogenesis was dose‐dependent (20 ng ‐ 2 μg/pellet), was prevented by anti‐TSP antibodies or by heat‐inactivation of TSP‐1, and was not due to inflammatory mediators, to FGF‐2 or to TGF‐β. Equimolar concentrations of the 25 kDa heparin binding fragment of TSP‐1 were even more efficient than the whole molecule, and promoted the angiogenic activity of FGF‐2. On the contrary, the 140 kDa fragment of TSP‐1 did not induce angiogenesis and turned off the angiogenic response to FGF‐2. The 25 kDa fragment and TSP‐1, but not the 140 kDa fragment, increased endothelial cell invasiveness and stimulated the production and activation of matrix metalloproteinase‐2 (MMP‐2). Moreover, the 25 kDa fragment reduced the synthesis of the MMP‐2 inhibitor TIMP‐2, while the 140 kDa fragment caused a twofold increase in TIMP‐2 production and inhibited MMPs stimulation by TSP‐1 and FGF‐2. We conclude that TSP‐1 is a source of smaller mediators of angiogenesis, which affect in an opposite way endothelial cell functions and proteolytic activity, thus resulting in an opposite final effect on angiogenesis.

Proliferation versus migration in platelet-derived growth factor signaling: the key role of endocytosis.

It is common knowledge that platelet-derived growth factor (PDGF) is a critical regulator of mesenchymal cell migration and proliferation. Nevertheless, these two cellular responses are mutually exclusive. To solve this apparent contradiction, we studied the behavior of NIH3T3 fibroblasts in response to increasing concentrations of PDGF. We found that there is strong cell proliferation induction only with PDGF concentrations >5 ng/ml, whereas the cell migration response arises starting from 1 ng/ml and is negligible at higher PDGF concentrations. According to these phenotypic evidences, our data indicate that cells display a differential activation of the main signaling pathways in response to PDGF as a function of the stimulation dose. At low PDGF concentrations, there is maximal activation of signaling pathways linked to cytoskeleton rearrangement needed for cell motility, whereas high PDGF concentrations activate pathways linked to mitogenesis induction. Our results suggest a mechanism by which cells switch from a migrating to a proliferating phenotype sensing the increasing gradient of PDGF. In addition, we propose that the cell decision to proliferate or migrate relies on different endocytotic routes of the PDGF receptor in response to different PDGF concentrations.

B1 receptor involvement in the effect of bradykinin on venular endothelial cell proliferation and potentiation of FGF‐2 effects

1 Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. 2 In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. 3 [3H]‐thymidine incorporation in subconfluent and synchronised CVEC was used to monitor DNA synthesis over 24 h. BK promoted a concentration‐dependent increase of DNA synthesis with maximal activity at 100 nm. At this concentration BK also induced 18 fold accumulation of c‐Fos protein immunoreactivity in the nucleus within 1 h from peptide exposure. 4 The total number of cells recovered after 48 h exposure to BK was increased in a concentration‐dependent manner. Maximal effect was produced by 100 nm concentration of the peptide which produced 50% increase in cell number. The selective B1 receptor agonist Des‐Arg9‐BK mimicked the proliferative effect of BK, while the B2 receptor agonist kallidin was devoid of any activity. The proliferation induced by BK was abolished in a concentration‐dependent manner by the addition of the B1 selective antagonist Des‐Arg9‐Leu8‐BK, while the selective B2 receptor antagonist HOE140 did not modify BK‐induced growth. 5 DNA synthesis and growth promoted by a threshold concentration of fibroblast growth factor‐2 (FGF‐2) (0.25 nm) were potentiated by increasing concentrations of BK and Des‐Arg9‐BK. 6 Endothelial cell migration assessed by the Boyden Chamber procedure was not promoted by BK or the selective B1 and B2 receptor agonists. 7 These data are the first demonstration that BK promotes growth of endothelial cells from postcapillary venules. The mitogenic activity of BK involves c‐Fos expression and potentiates the growth promoting effect of FGF‐2. Only the B1 receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules.

Effect of hypoxia and endothelial loss on vascular smooth muscle cell responsiveness to VEGF-A: role of flt-1/VEGF-receptor-1

OBJECTIVE The influence of hypoxia and endothelial loss on the responsiveness of vascular smooth muscle cells (VSMCs) to vascular endothelial growth factor (VEGF-A) was tested. METHODS AND RESULTS Exposure to hypoxia induced a potentiation of cultured cell proliferation in response to either the agonist for the VEGF receptor 1 (flt-1) placental growth factor (PlGF-1) or to VEGF-A. This effect was mediated by the mitogen activated protein kinase (MAPK) cascade, since it was inhibited by the MAPK kinase inhibitor PD98059 and by the farnesyl transferase inhibitor II. Accordingly, PlGF-1 activated extracellular signal-regulated kinase(1/2). In rat aortic rings deprived of endothelium and cultured in three-dimensional fibrin gels, an increased sprouting of tubular structures in response to VEGF-A was observed only under hypoxia. Studies on rat aorta preparations revealed an endothelium-dependent vasorelaxation in response to either VEGF-A or PlGF1, which was reversed to a contractile response in endothelium-deprived preparations exposed to hypoxia. Western blot and immunohistochemistry of endothelium-deprived preparations exposed to hypoxia showed flt-1 receptor expression in all medial cells. Conversely, flt-1 mRNA, of endothelium-deprived aortic preparations and of tubular structures, was unchanged by hypoxia. CONCLUSION These findings demonstrate that experimental conditions mimicking pathological vascular injury can make VSMCs responsive to VEGF-A through the induction of functional flt-1 receptors.

Intracellular pathways triggered by the selective FLT-1-agonist placental growth factor in vascular smooth muscle cells exposed to hypoxia

We have previously shown that hypoxia makes vascular smooth muscle cells (VSMCs) responsive to placental growth factor (P1GF) through the induction of functional fms‐like tyrosine kinase (Flt‐1) receptors. The aim of this study was to investigate the molecular mechanisms involved in the P1GF effects on proliferation and contraction of VSMCs previously exposed to hypoxia (3% O2). In cultured rat VSMCs exposed to hypoxia, P1GF increased the phosphorylation of protein kinase B (Akt), p38 and STAT3; activation of STAT3 was higher than that of other kinases. In agreement with this finding, the proliferation of hypoxia‐treated VSMCs in response to P1GF was significantly impaired by the p38 and the phosphatidylinositol 3‐kinase inhibitors SB202190 and LY294002, respectively, and was almost completely prevented by AG490, a janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) inhibitor. Since hypoxia was able to reverse the vasorelaxant effect of P1GF into a vasoconstrictor response, the mechanism of this latter effect was also investigated. Significant Flt‐1 activity was measured in isolated preparations from rat aorta exposed to hypoxia. Inhibitors of mitogen‐activated protein kinase kinase, Akt and STAT3 induced a modest inhibition of the vasoconstrictor response to P1GF, while the p38 inhibitor SB202190 markedly impaired the P1GF‐induced contractile response. These effects were selectively mediated by Flt‐1 without any involvement of foetal liver kinase‐1 receptors. These data are the first evidence that different intracellular pathways activated by Flt‐1 receptor in VSMCs are involved in diverse biological effects of P1GF: while mitogen activated protein kinase kinase/extracellular signal regulated kinase1/2 and JAK/STAT play a role in VSMC proliferation, p38 is involved in VSMC contraction. These findings may highlight the role of P1GF in vascular pathology.

The tachykinin NK1 receptor mediates the migration-promoting effect of substance P on human skin fibroblasts in culture

Fibroblast migration is an important component of the tissue response during the repair process, and substance P (SP) has been shown to exert trophic effects. In the present study, cell migration was evaluated as the distance travelled by adherent human skin fibroblasts (HF) at 96 h and by the number of individual cells moving across a filter within 5 h.In control conditions (1% calf serum) adherent fibroblasts moved from the starting line by approximately 700 μm. The addition of SP (10−11–10−7 M) increased HF mobilisation in a concentration-dependent manner, with maximal activity at 10−8 M (50% increase in migration over control). Migration of individual HF in suspension was also promoted by SP in a concentration-dependent manner, with an EC50 of 2.2×10−9 M. The response produced by the maximally effective concentration of SP was equal to 65 and 90% of the effect elicited by 100 ng/ml Platelet-Derived Growth Factor AB (PDGF A/B) on adherent and individual cells respectively. The synthetic NK1 receptor agonist [Sar9]SP-sulphone (10−11–10−6 M) reproduced the SP effect. The NK2 and NK3 receptor agonists [βAla8]NKA(4–10) and [MePhe7]NKB were devoid of any effect. The effect of SP was antagonised by two selective antagonists of NK1 receptors, namely (±) CP 96,345 (10−10–10−8 M) and FK 888 (10−9–10−7 M), while the NK2 receptor antagonist MEN 10627 (10−8–10−7 M) was not effective.Our data indicate that SP is a potent effector of fibroblast migration and the NK1 receptor is responsible for this effect. These observations further support the specific role of the NK1 receptor in mediating the trophic function of SP at the cutaneous level.

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells.

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.

What is the evidence for the role of TRP channels in inflammatory and immune cells

A complex network of many interacting mechanisms orchestrates immune and inflammatory responses. Among these, the cation channels of the transient receptor potential (TRP) family expressed by resident tissue cells, inflammatory and immune cells and distinct subsets of primary sensory neurons, have emerged as a novel and interrelated system to detect and respond to harmful agents. TRP channels, by means of their direct effect on the intracellular levels of cations and/or through the indirect modulation of a large series of intracellular pathways, orchestrate a range of cellular processes, such as cytokine production, cell differentiation and cytotoxicity. The contribution of TRP channels to the transition of inflammation and immune responses from a defensive early response to a chronic and pathological condition is also emerging as a possible underlying mechanism in various diseases. This review discusses the roles of TRP channels in inflammatory and immune cell function and provides an overview of the effects of inflammatory and immune TRP channels on the pathogenesis of human diseases.