Prolyl oligopeptidase induces angiogenesis both in vitro and in vivo in a novel regulatory manner.

BACKGROUND AND PURPOSE A serine protease, prolyl oligopeptidase (POP) has been reported to be involved in the release of the pro-angiogenic tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (Ac-SDKP) from its precursor, 43-mer thymosin ß4 (Tß4). Recently, it was shown that both POP activity and the levels of Ac-SDKP are increased in malignant tumours. The aim of this study was to clarify the release of Ac-SDKP, and test if POP and a POP inhibitor, 4-phenyl-butanoyl-L-prolyl-2(S)-cyanopyrrolidine (KYP-2047), can affect angiogenesis. EXPERIMENTAL APPROACH We used HPLC for bioanalytical and an enzyme immunoassay for pharmacological analysis. Angiogenesis of human umbilical vein endothelial cells was assessed in vitro using a 'tube formation' assay and in vivo using a Matrigel plug assay (BD Biosciences, San Jose, CA, USA) in adult male rats. Moreover, co-localization of POP and blood vessels was studied. KEY RESULTS We showed the sequential hydrolysis of Tß4: the first-step hydrolysis by proteases to <30-mer peptides is followed by an action of POP. Unexpectedly, POP inhibited the first hydrolysis step, revealing a novel regulation system. POP with Tß4 significantly induced, while KYP-2047 effectively prevented, angiogenesis in both models compared with Tß4 addition itself. POP and endothelial cells were abundantly co-localized in vivo. CONCLUSIONS AND IMPLICATIONS We have now revealed that POP is a second-step enzyme in the release of Ac-SDKP from Tß4, and it has novel autoregulatory effect in the first step. Our results also advocate a role for Ac-SDKP in angiogenesis, and suggest that POP has a pro-angiogenic role via the release of Ac-SDKP from its precursor Tß4 and POP inhibitors can block this action.

Expression and traffic of cellular prolyl oligopeptidase are regulated during cerebellar granule cell differentiation, maturation, and aging.

Prolyl oligopeptidase (POP) is an endopeptidase which cleaves short proline-containing neuropeptides, and it is involved in memory and learning. POP also has an intercellular function mediated through the inositol pathway, and has been involved in cell death. POP has been early considered as a housekeeping enzyme, but the recent research indicates that POP expression is regulated across tissues and intracellularly. In the brain, POP is exclusively expressed in neurons and most abundantly in pyramidal neurons of cerebral cortex, in the CA1 field neurons of hippocampus and in cerebellar Purkinje's cells. Intracellularly, POP is mainly present in the cytoplasm and some in intracellular membranes, like rough endoplasmic reticulum and Golgi apparatus. In this paper, we systematically studied the levels of expression of POP along the life of cerebellar granule cells (CGC) in culture and the distribution of POP within different intracellular compartments. We used the tight-binding inhibitor JTP-4819 covalently coupled with fluorescein (FJTP) as a tool to study the changes on expression and localization of POP protein. Our results indicate that POP activity levels are regulated during the life of the neurons. POP was found mainly in cytoplasm and neuronal projections, but at an early developmental phase significant amounts were found also in nuclei. Along the life of the neurons, POP activity fluctuated in 7-day cycles. In young neurons, the cytosolic POP activity was low but increased by maturation so that the activity peak coincided with full differentiation. Over aging, cytoplasmic POP was concentrated around nucleus, but the activity decreased with time. POP was also present in vesicles across the neuron. No major changes were seen in the nuclear or membrane bound POP over aging until activity disappeared upon neuronal death. This is the first time when POP was found in the nuclei of human neuronal cells.

A risk model for non-small cell lung cancer using clinicopathological variables, angiogenesis and oncoprotein expression.

BACKGROUND: The aim of this study was to investigate new prognostic factors, by using a prognostic model, that could help to identify the patient group with the greatest probability of death. PATIENTS AND METHODS: First, the clinicopathological variables were analyzed. Second, microvessels were immunohistochemically (IHC) stained with factor VIII-related antibody and then counted in the most intense vascularization area or hotspot, using an automatic image analyzer. In addition, biological angiogenesis-related factors such as vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase expression (iNOS) were also studied. Finally, we evaluated the IHC expression of p53 and p21WAF1 tumor supressor proteins. RESULTS: The significant independent predictors were: tumor size (p=0.0063), angiogenesis (p=0.0271) and p21WAF1 (p=0.0478). Thus, the most important factor was tumor size 2.7462 [95% CI=1.3307-5.6673]. Finally, these variables were included in a risk model, in order to identify the group with the highest associated probability of death. CONCLUSION: The analysis of several prognostic factors could establish a more accurate patient risk profile.

Polymerase chain reaction detection of BK virus and monitoring of BK nephropathy in renal transplant recipients at the University Hospital La Fe.

INTRODUCTION: Reactivation of BK infection occurs in immunocompromised hosts causing tubulointerstitial nephropathy (BKVN). Approximately 5% of kidney transplant recipients (KTR) develop BKVN, special half of whom lose their grafts. However, BKVN morphologic diagnosis on a renal biopsy is complicated, because the cytopathic changes can sometimes mimic rejection. Thus, BKV DNA-polymerase chain reaction (PCR) assay on serum, urine, and renal tissue is useful for early detection and monitoring of BKV. MATERIALS AND METHODS: We performed routine monthly urine cytologies looking for decoy cells as a marker of virus replication. Then, we performed a qualitative PCR on urine and serum in all recipients (independently of positive or negative cytology). We amplified 3 BK viral genome regions, LT (early transcription region) and VP1 (late transcription region) seeking a more accurate virus detection, and the TCR (control transcription region) region to perform a polymorphism sequence analysis to identify the BK genomic variant. Finally, the BKVN diagnosis was confirmed using renal biopsy. RESULTS: At present, 132 patients have been monitored. Thirteen of 40 (33%) were PCR-urine-positive cases (5 LT+/VP1- and 8 LT+/VP1+), and 10 of 132 (7.5%) were PCR-serum-positive cases (7 LT+/VP1- and 3 LT+/VP1+). When we compared PCR-urine and cytology results, 11 of 40 (27.5%) patients showed a positive cytology, 6 of whom were PCR- urine-positive (1 LT+/VP1- and 5 LT+/VP1+); whereas, 29 patients showed a negative cytology, 7 of whom were PCR-urine-positive(3 LT+/VP1- and 4 LT+/VP1+). Thus, comparison of PCR- urine and cytology results revealed false-positive and false-negative cases. Finally, TCR sequence analysis was performed in 9 patients to identify the BK genomic variants. CONCLUSION: Testing for BKV DNA in urine and serum is a noninvasive early detection assay and monitoring tool.