Giordano Pula

Proteomics identifies thymidine phosphorylase as a key regulator of the angiogenic potential of colony-forming units and endothelial progenitor cell cultures.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-d-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin &agr;v&bgr;3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

Agonist-induced internalization of the metabotropic glutamate receptor 1a is arrestin- and dynamin-dependent

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N‐terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA‐mGluR1a, the epitope‐tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 µm; 30 min) the HA‐mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist‐induced internalization of the HA‐mGluR1a. To determine whether agonist‐induced mGluR1a internalization is an arrestin‐ and dynamin‐dependent process, cells were cotransfected with HA‐mGluR1a and either of these dynamin‐K44A or arrestin‐2 (319–418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate‐induced (10 µm; 30 min) HA‐mGluR1a internalization. In addition, wild‐type arrestin‐2−green fluorescent protein (arrestin‐2−GFP) or arrestin‐3−GFP underwent agonist‐induced translocation from cytosol to membrane in HEK293 cells coexpressing HA‐mGluR1a. Taken together our observations demonstrate that agonist‐induced internalization of mGluR1a is an arrestin‐ and dynamin‐dependent process.

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets

VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser157 by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser157, but not Ser239, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser157, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser157 VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser157 in response to thrombin in platelets.

Pharmacological characterisation of the human small conductance calcium-activated potassium channel hSK3 reveals sensitivity to tricyclic antidepressants and antipsychotic phenothiazines.

A stable CHO-K1 cell line was developed which expresses the human small conductance calcium-activated potassium channel hSK3. Immunofluorescence microscopy using an anti-SK3 antibody and radioligand binding using [(125)I]-apamin demonstrated the presence of hSK3 channel in the recombinant cell line. This cell line was utilised in a fluorescence assay using the membrane potential-sensitive dye DiBAC(4)(3) to functionally analyse and pharmacologically characterise this potassium channel. The analysis of known blockers of calcium-activated potassium channels revealed the highest potency for apamin (IC(50)=13.2 nM). This result was confirmed by direct recordings of SK3 currents using the whole-cell patch-clamp technique. Tricyclic antidepressants such as desipramine, imipramine and nortriptyline as well as phenothiazines such as fluphenazine, promethazine, chlorpromazine and trifluoperazine blocked the hSK3 channel with micromolar potencies. These compounds also displaced [(125)I]-apamin binding to the hSK3 channel thus suggesting direct and competitive channel blocking activity. Since these compounds share a common three-ring molecular core structure, this feature seems to be important for channel blocking activity. The serine/threonine protein phosphatase inhibitors okadaic acid and calyculin A were able to abolish channel activation with nanomolar potencies, but did not displace [(125)I]-apamin binding. Thus, phosphorylation of hSK3 or an accessory channel subunit seems to be involved in its modulation.

Functional interaction of protein kinase Cα with the tyrosine kinases Syk and Src in human platelets

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCα, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Gö6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCα but not PKCβ was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCα associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCα and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCα is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKCα, consistent with a lack of effect of PKCα inhibition on Syk activity. PKCα also associates with Src; although in contrast to interaction with Syk, PKCα activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Gö6976. Therefore, there is a complex interplay between PKCα, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.

Reactive Oxygen Species: Physiological Roles in the Regulation of Vascular Cells

Reactive oxygen species (ROS) are now appreciated to play several important roles in a number of biological processes and regulate cell physiology and function. ROS are a heterogeneous chemical class that includes radicals, such as superoxide ion (O2(•-)), hydroxyl radical (OH(•)) and nitric oxide (NO(•)), and non-radicals, such as hydrogen peroxide (H2O2), singlet oxygen ((1)O2), hypochlorous acid (HOCl), and peroxynitrite (NO3 (-)). In the cardiovascular system, besides playing a critical role in the development and progression of vasculopathies and other important pathologies such as congestive heart failure, atherosclerosis and thrombosis, ROS also regulate physiological processes. Evidence from a wealth of cardiovascular research studies suggests that ROS act as second messengers and play an essential role in vascular homeostasis by influencing discrete signal transduction pathways in various systems and cell types. They are produced throughout the vascular system, regulate differentiation and contractility of vascular smooth muscle cells, control vascular endothelial cell proliferation and migration, mediate platelet activation and haemostasis, and significantly contribute to the immune response. Our understanding of ROS chemistry and cell biology has evolved to the point of realizing that different ROS have distinct and important roles in cardiovascular physiology. This review will outline sources, functions and molecular mechanisms of action of different ROS in the cardiovascular system and will describe their emerging role in healthy cardiovascular physiology and homeostasis.

Agonist‐independent internalization of metabotropic glutamate receptor 1a is arrestin‐ and clathrin‐dependent and is suppressed by receptor inverse agonists

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36‐7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate‐induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36‐7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36‐7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36‐7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin‐2 [arrestin‐2‐(319–418)] or Eps15 [Eps15(EΔ95‐295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36‐7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin‐ and clathrin‐dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

The novel NOX inhibitor 2‐acetylphenothiazine impairs collagen‐dependent thrombus formation in a GPVI‐dependent manner

NADPH oxidases (NOXs) contribute to platelet activation by a largely unknown mechanism. Here, we studied the effect of the novel NOX inhibitor 2‐acetylphenothiazine (2‐APT) on human platelet functional responses and intracellular signaling pathways.

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization

To investigate the role of the intracellular C‐terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C‐terminal tail deletion mutants (Arg847stop, DM‐I; Arg868stop, DM‐II; Val893stop, DM‐III). Quantification of glutamate‐induced internalization provided by ELISA indicated that DM‐III, like the wild‐type mGlu1a, underwent rapid internalization whilst internalization of DM‐I and DM‐II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate‐induced mGlu1a internalization, had no effect on the internalization of DM‐I, DM‐II, or DM‐III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC‐ and Ca2+‐calmodulin‐dependent protein kinase II‐dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co‐expression of a dominant negative mutant form of G protein‐coupled receptor kinase 2 (DNM‐GRK2; Lys220Arg) significantly attenuated glutamate‐induced internalization of mGlu1a and DM‐III, whilst internalization of DM‐I and DM‐II was not significantly affected. The glutamate‐induced internalization of mGlu1a and DM‐III, but not of DM‐I or DM‐II, was inhibited by expression of DNM‐arrestin [arrestin‐2(319–418)]. In addition glutamate‐induced rapid translocation of arrestin‐2‐Green Fluorescent Protein (arr‐2‐GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM‐III. Functionally, in cells expressing mGlu1a, glutamate‐stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM‐II and DM‐III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894–Leu1199), whilst in contrast glutamate‐stimulated GRK‐ and arrestin‐dependent regulation of this receptor depends on a region of 25 amino acids (Ser869–Val893) in the proximal C‐terminal tail.

Role of amyloid peptides in vascular dysfunction and platelet dysregulation in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative cause of dementia in the elderly. AD is accompanied by the accumulation of amyloid peptides in the brain parenchyma and in the cerebral vessels. The sporadic form of AD accounts for about 95% of all cases. It is characterized by a late onset, typically after the age of 65, with a complex and still poorly understood aetiology. Several observations point towards a central role of cerebrovascular dysfunction in the onset of sporadic AD (SAD). According to the “vascular hypothesis”, AD may be initiated by vascular dysfunctions that precede and promote the neurodegenerative process. In accordance to this, AD patients show increased hemorrhagic or ischemic stroke risks. It is now clear that multiple bidirectional connections exist between AD and cerebrovascular disease, and in this new scenario, the effect of amyloid peptides on vascular cells and blood platelets appear to be central to AD. In this review, we analyze the effect of amyloid peptides on vascular function and platelet activation and its contribution to the cerebrovascular pathology associated with AD and the progression of this disease.