Identification of aspartic acid-203 in human thymidine phosphorylase as an important residue for both catalysis and non-competitive inhibition by the small molecule "crystallization chaperone" 5'-O-tritylinosine (KIN59)

Thymidine phosphorylase (TP) is a catabolic enzyme in thymidine metabolism that is frequently upregulated in many solid tumors. Elevated TP levels are associated with tumor angiogenesis, metastasis and poor prognosis. Therefore, the use of TP inhibitors might offer a promising strategy for cancer treatment. The tritylated inosine derivative 5'-O-tritylinosine (previously designated KIN59) is a noncompetitive inhibitor of TP which was previously found to be instrumental for the crystallization of human TP. A combination of computational studies including normal mode analysis, automated ligand docking and molecular dynamics simulations were performed to define a plausible binding site for 5'-O-tritylinosine on human TP. A cavity in which 5'-O-tritylinosine could fit was identified in the vicinity of the Gly405-WI419 loop at a distance of about 11 angstrom from the substrate-binding site. In the X-ray crystal structure, this pocket is characterized by an intricate hydrogen-bonding network in which Asp203 was found to play an important role to afford the loop stabilization that is required for efficient enzyme catalysis. Site-directed mutagenesis of this amino acid residue afforded a mutant enzyme with a severely compromised catalytic efficiency (V-max /K-m of mutant enzyme similar to 50-fold lower than for wild-type TP) and pronounced resistance to the inhibitory effect of 5'-O-tritylinosine. In contrast, the D203A mutant enzyme kept full sensitivity to the competitive inhibitors 6-aminothymine and 6-amino-5-bromouracil, which is in line with the kinetic properties of these inhibitors. Our findings reveal the existence of a previously unrecognized site in TP that can be targeted by small molecules to inhibit the catalytic activity of TP. (C) 2009 Elsevier Inc. All rights reserved

The COOH-Terminal Peptide of Platelet Factor-4 Variant (CXCL4L1/PF-4var47-70) Strongly Inhibits Angiogenesis and Suppresses B16 Melanoma Growth In vivo.

Chemokines influence tumor growth directly or indirectly via both angiogenesis and tumor-leukocyte interactions. Platelet factor-4 (CXCL4/PF-4), which is released from alpha-granules of activated platelets, is the first described angiostatic chemokine. Recently, it was found that the variant of CXCL4/PF-4 (CXCL4L1/PF-4var) could exert a more pronounced angiostatic and antitumoral effect than CXCL4/PF-4. However, the molecular mechanisms of the angiostatic activities of the PF-4 forms remain partially elusive. Here, we studied the biological properties of the chemically synthesized COOH-terminal peptides of CXCL4/PF-4 (CXCL4/PF-4(47-70)) and CXCL4L1/PF-4var (CXCL4L1/PF-4var(47-70)). Both PF-4 peptides lacked monocyte and lymphocyte chemotactic activity but equally well inhibited (25 nmol/L) endothelial cell motility and proliferation in the presence of a single stimulus (i.e., exogenous recombinant fibroblast growth factor-2). In contrast, when assayed in more complex angiogenesis test systems characterized by the presence of multiple mediators, including in vitro wound-healing (2.5 nmol/L versus 12.5 nmol/L), Matrigel (60 nmol/L versus 300 nmol/L), and chorioallantoic membrane assays, CXCL4L1/PF-4var(47-70) was found to be significantly (5-fold) more angiostatic than CXCL4/PF-4(47-70). In addition, low (7 mug total) doses of intratumoral CXCL4L1/PF-4var(47-70) inhibited B16 melanoma growth in mice more extensively than CXCL4/PF-4(47-70). This antitumoral activity was predominantly mediated through inhibition of angiogenesis (without affecting blood vessel stability) and induction of apoptosis, as evidenced by immunohistochemical and fluorescent staining of B16 tumor tissue. In conclusion, CXCL4L1/PF-4var(47-70) is a potent antitumoral and antiangiogenic peptide. These results may represent the basis for the design of CXCL4L1/PF-4var COOH-terminal-derived peptidomimetic anticancer drugs. Mol Cancer Res; 8(3); 322-34

Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct <i>in vitro</i>

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury

Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

BACKGROUND: Thymidine phosphorylase (TP) is often overexpressed in tumours and has a role in tumour aggressiveness and angiogenesis. Here, we determined whether TP increased tumour invasion and whether TP-expressing cancer cells stimulated angiogenesis. METHODS: Angiogenesis was studied by exposing endothelial cells (HUVECs) to conditioned medium (CM) derived from cancer cells with high (Colo320TP1 = CT-CM, RT112/TP = RT-CM) and no TP expression after which migration (wound-healing-assay) and invasion (transwell-assay) were determined. The involvement of several angiogenic factors were examined by RT-PCR, ELISA and blocking antibodies. RESULTS: Tumour invasion was not dependent on intrinsic TP expression. The CT-CM and RT-CM stimulated HUVEC-migration and invasion by about 15 and 40%, respectively. Inhibition by 10 mu M TPI and 100 mu M L-dR, blocked migration and reduced the invasion by 50-70%. Thymidine phosphorylase activity in HUVECs was increased by CT-CM. Reverse transcription-polymerase chain reaction revealed a higher mRNA expression of bFGF (Colo320TP1), IL-8 (RT112/TP) and TNF-alpha, but not VEGF. Blocking antibodies targeting these factors decreased the migration and invasion that was induced by the CT-CM and RT-CM, except for IL-8 in CT-CM and bFGF in RT-CM. CONCLUSION: In our cell line panels, TP did not increase the tumour invasion, but stimulated the migration and invasion of HUVECs by two different mechanisms. Hence, TP targeting seems to provide a potential additional strategy in the field of anti-angiogenic therapy

The thymidine phosphorylase inhibitor 5′-O-tritylinosine (KIN59) is an antiangiogenic multitarget fibroblast growth factor-2 antagonist

5′-O-Tritylinosine (KIN59) is an allosteric inhibitor of the angiogenic enzyme thymidine phosphorylase. Previous observations showed the capacity of KIN59 to abrogate thymidine phosphorylase-induced as well as developmental angiogenesis in the chicken chorioallantoic membrane (CAM) assay. Here, we show that KIN59 also inhibits the angiogenic response triggered by fibroblast growth factor-2 (FGF2) but not by VEGF in the CAM assay. Immunohistochemical and reverse transcriptase PCR analyses revealed that the expression of laminin, the major proteoglycan of the basement membrane of blood vessels, is downregulated by KIN59 administration in control as well as in thymidine phosphorylase- or FGF2-treated CAMs, but not in CAMs treated with VEGF. Also, KIN59 abrogated FGF2-induced endothelial cell proliferation, FGF receptor activation, and Akt signaling in vitro with no effect on VEGF-stimulated biologic responses. Accordingly, KIN59 inhibited the binding of FGF2 to FGF receptor-1 (FGFR1), thus preventing the formation of productive heparan sulphate proteoglycan/FGF2/FGFR1 ternary complexes, without affecting heparin interaction. In keeping with these observations, systemic administration of KIN59 inhibited the growth and neovascularization of subcutaneous tumors induced by FGF2-transformed endothelial cells injected in immunodeficient nude mice. Taken together, the data indicate that the thymidine phosphorylase inhibitor KIN59 is endowed with a significant FGF2 antagonist activity, thus representing a promising lead compound for the design of multi-targeted antiangiogenic cancer drugs. ©2012 AACR.Peer Reviewe

Isolation and characterization of equine native MSC populations

Abstract Background In contrast to humans in which mesenchymal stem/stromal cell (MSC) therapies are still largely in the clinical trial phase, MSCs have been used therapeutically in horses for over 15 years, thus constituting a valuable preclinical model for humans. In human tissues, MSCs have been shown to originate from perivascular cells, namely pericytes and adventitial cells, which are identified by the presence of the cell surface markers CD146 and CD34, respectively. In contrast, the origin of MSCs in equine tissues has not been established, preventing the isolation and culture of defined cell populations in that species. Moreover, a comparison between perivascular CD146+ and CD34+ cell populations has not been performed in any species. Methods Immunohistochemistry was used to identify adventitial cells (CD34+) and pericytes (CD146+) and to determine their localization in relation to MSCs in equine tissues. Isolation of CD34+ (CD34+/CD146–/CD144–/CD45–) and CD146+ (CD146+/CD34–/CD144–/CD45–) cell fractions from equine adipose tissue was achieved by fluorescence-activated cell sorting. The isolated cell fractions were cultured and analyzed for the expression of MSC markers, using qPCR and flow cytometry, and for the ability to undergo trilineage differentiation. Angiogenic properties were analyzed in vivo using a chorioallantoic membrane (CAM) assay. Results Both CD34+ and CD146+ cells displayed typical MSC features, namely growth in uncoated tissue culture dishes, clonal growth when seeded at low density, expression of typical MSC markers, and multipotency shown by the capacity for trilineage differentiation. Of note, CD146+ cells were distinctly angiogenic compared with CD34+ and non-sorted cells (conventional MSCs), demonstrated by the induction of blood vessels in a CAM assay, expression of elevated levels of VEGFA and ANGPT1, and association with vascular networks in cocultures with endothelial cells, indicating that CD146+ cells maintain a pericyte phenotype in culture. Conclusion This study reports for the first time the successful isolation and culture of CD146+ and CD34+ cell populations from equine tissues. Characterization of these cells evidenced their distinct properties and MSC-like phenotype, and identified CD146+ cells as distinctly angiogenic, which may provide a novel source for enhanced regenerative therapies

What's the effect of the implementation of general practitioner cooperatives on caseload? Prospective intervention study on primary and secondary care

<p>Abstract</p> <p>Background</p> <p>Out-of-hours care in the primary care setting is rapidly changing and evolving towards general practitioner 'cooperatives' (GPC). GPCs already exist in the Netherlands, the United Kingdom and Scandinavia, all countries with strong general practice, including gatekeepers' role. This intervention study reports the use and caseload of out-of-hours care before and after implementation of a GPC in a well subscribed region in a country with an open access health care system and no gatekeepers' role for general practice.</p> <p>Methods</p> <p>We used a prospective before/after interventional study design. The intervention was the implementation of a GPC.</p> <p>Results</p> <p>One year after the implementation of a GPC, the number of patient contacts in the intervention region significantly increased at the GPC (OR: 1.645; 95% CI: 1.439-1.880), while there were no significant changes in patient contacts at the Emergency Department (ED) or in other regions where a simultaneous registration was performed. Although home visits decreased in all general practitioner registrations, the difference was more pronounced in the intervention region (intervention region: OR: 0.515; 95% CI: 0.411-0.646, other regions: OR: 0.743; 95% CI: 0.608-0.908). At the ED we observed a decrease in the number of trauma cases (OR: 0.789; 95% CI: 0.648-0.960) and of patients who came to hospital by ambulance (OR: 0.687; 95% CI: 0.565-0.836).</p> <p>Conclusions</p> <p>One year after its implementation more people seek help at the GPC, while the number of contacts at the ED remains the same. The most prominent changes in caseload are found in the trauma cases. Establishing a GPC in an open health care system, might redirect some patients with particular medical problems to primary care. This could lead to a lowering of costs or a more cost-effective out of hours care, but further research should focus on effective usage to divert patient flows and on quality and outcome of care.</p

Mesenchymal stem cell-based therapy for ischemic stroke

Ischemic stroke represents a major, worldwide health burden with increasing incidence. Patients affected by ischemic strokes currently have few clinically approved treatment options available. Most currently approved treatments for ischemic stroke have narrow therapeutic windows, severely limiting the number of patients able to be treated. Mesenchymal stem cells represent a promising novel treatment for ischemic stroke. Numerous studies have demonstrated that mesenchymal stem cells functionally improve outcomes in rodent models of ischemic stroke. Recent studies have also shown that exosomes secreted by mesenchymal stem cells mediate much of this effect. In the present review, we summarize the current literature on the use of mesenchymal stem cells to treat ischemic stroke. Further studies investigating the mechanisms underlying mesenchymal stem cells tissue healing effects are warranted and would be of benefit to the field