Prognostic role of tumor necrosis, microvessel density, vascular endothelial growth factor and hypoxia inducible factor-1alpha in patients with clear cell renal carcinoma after radical nephrectomy in a long term follow-up.

Angiogenesis is a critical step in the growth, invasive progression and metastatic spread of solid tumors. We investigated the importance of tumor necrosis, and microvessel density (MVD), vascular endothelial growth factor (VEGF) and hypoxia inducible factor 1α (HIF-1α) immunohistochemical expression in a large series of clear cell renal carcinomas treated with radical nephrectomy and assessed the prognostic value of their expression in terms of patient survival at long-term follow up. Fifty patients with clear cell RCC were examined. The features considered when evaluating the patients were age, tumor size and grade, intratumoral vascular and renal capsula invasion, histological necrosis, and MVD, vascular and tumoral cell VEGF, and vascular, tumoral cytoplasmic and nuclear HIF-1α expression on the histologic specimens. All considered parameters were correlated with patient specific survival. Mean age was 62.06 ± 6.8 years. Median follow-up was 191.66 months; median survival was 120.86 months. Twenty-one patients developed metastases in the follow-up. Tumor necrosis, microvascular invasion and renal capsula infiltration are more likely to occur in high stage and grade RCC; cytoplasmic HIF-1α is highly expressed in high grade RCC. Survival is dependent upon tumor stage and grade, the presence of intratumoral vascular invasion and capsular infiltration, and tumor necrosis; MVD also resulted as being an important prognostic factor. VEGF and HIF-1α correlate with prognosis in high stage tumors where VEGF is the most important independent prognostic factor for cancer specific death. The histological and immunohistochemical parameters considered in our study can influence disease recurrence and survival in RCC

IGF1 and IGF1/VEGF cross-talk on human mesenchymal stromal cells (hMSCs): the role of stem cell sources in bone healing

Repair of skeletal defects remains a considerable biomedical problem. One of the major obstacles of the different tested strategies still remains the vascularization of engineered scaffolds. To this aim we have examined the ability of IGF1, alone or in association with VEGF, to modulate Periosteum Derived Progenitor Cells (PDPCs) (Ferretti et al., 2012) and Skin-Mesenchymal Stromal Cells (S-MSCs) (Orciani and Di Primio, 2013) osteoblastic or endothelial commitment. A selected gene panel for endothelial and osteoblastic differentiation as well as genes that can affect MAPK and PI3K/AKT signalling pathways were investigated. Our results showed a different commitment of PDPCs and S-MSCs under growth factor stimulation: the former are induced towards an osteoblastic differentiation, whilst the latter seem to be brought to an endothelial phenotype. This commitment is also related to a diverse MAPK or PI3K/AKT signalling pathway activation. Our results open intriguing perspective for the development of innovative bone tissue engineering approaches based on associated angiogenesis and osteogenesis. Further investigations are however necessary to gain insight on the real cross-talk between proliferation and differentiation in adult stem cells. The work was supported by Italian FIRB (RBAP10MLK7) and PRIN (2010J8RYS7) project grants

Loss of nuclear BAP1 protein expression is a marker of poor prognosis in patients with clear cell renal cell carcinoma

BAP1 is a gene situated on chromosome 3p in a region that is deleted in over 90% of Renal Cell Carcinomas (RCCs) (1,2). In the present study we studied BAP1 immunohistochemical expression in a large series of conventional clear cell RCCs (ccRCCs) treated with radical nephrectomy and we assessed the prognostic value of their expression in terms of patients survival at long-term follow-up. 154 consecutive patients with ccRCC were selected from a prospective database and considered for the study purpose; all patients were treated with radical nephrectomy and lymphadenectomy at our Institute of Urology between 1983 and 1985. The features considered in this study were tumor size, grade and stage, vascular and capsular invasion, incidence of metastasis and patient specific survival; all these parameters were correlated with immunohistochemical cytoplasmic and nuclear expression of BPA-1 in tumoral tissue. Median follow-up was 196.18 months (range 5 to 274); median survival was 125.34 months (range 5 to 274 months). We found that nuclear BAP1 expression showed a high frequency of loss in tumoral cells; nuclear BAP1 negative tumors had higher tumor size, higher Fuhrman grade, and higher stage, a greater amount of vascular and capsular invasion and a higher incidence of metastases. We have demonstrated that nuclear BAP1 expression is a marker of prognosis in ccRCC, having an impact on cancer-specific survival. The clinical importance for BAP1 will be realized with the identification and application of targeted therapies and with individualized approaches in the adjuvant and/or metastatic setting

In vitro biological effects of raw and thermally treated asbestos-containing materials

Asbestos cement, the main asbestos-containing material (ACM) manufactured in Italy in the past, is a health hazard whose elimination is a priority concern. Asbestos fibers can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation. In this study human A549 cells were directly exposed to raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 °C (HT-CA) and raw commercial grey cement (GC) for 24 and 48h, or treated with conditioned culture medium up to 96 h. In our previous studies we demonstrated that the final product of heat treatment of cement asbestos was considerably more inert and had lower cytotoxic potential than the original asbestos material. However, to better evaluate the risks of interactions with the materials, further in vitro investigations were performed concerning fiber-cell superficial interactions, immuno-hystochemical expression of cytochines p53, p53 homologue p73, TNF-related apoptosis- inducing ligand (TRAIL), and conditioned medium effects on cell viability. Data showed more severe cytotoxic damage by raw cement-asbestos compared to the heat treated materials and different expressions of cytochines that exert critical role in regulating the cell response to asbestos-induced DNA damage. These data should be taken in consideration for a safe recycling of thermal transformed asbestos materials

Bone regeneration strategies in the elderly: the role of ageing and replicative senescence in periosteal-derived stem cells

Periosteum contains resident progenitor cells (PDPCs) representing an attractive alternative source of mesenchymal stem cells (MSCs) for skeletal tissue engineering approaches based on cell recruitment (1). Increased in life expectancy point out the necessity for customized strategies to restore bone loss due to trauma and/or disease in elderly. Aim of the present research was the evaluation of the ageing impact on PDPCs isolated from differently aged subjects. Moreover, since long-term culture could lead MSCs to senescence, the effects of culture expansion method on young PDPC through sequential serial passages were examined. Age-related increase of p53 expression and impairment in proliferating capacity were observed; those findings were strictly related to nitric oxide (NO) release. Moreover, qRT-PCR analysis showed a greater expression of genes involved in bone remodelling in elderly donors. As far as replicative in vitro expansion was concerned, we observed that later PDPC passages exhibited the typical “replicative senescence” features (i.e. flattened and enlarged morphology, prolonged population doubling time and increased SA-βgal activity). In these cells, p16 rather than p53 seemed to be involved in senescence processes. Similarly to the elderly, the decrease in proliferating ability of in vitro senescent PDPCs was concomitant with a higher NO production, and the changes in the expression of genes involved in bone resorption and RANKL/OPG ratio were superimposable. Interestingly, the relationship between NO release and ageing could represent a cutting edge “replicative senescence index” as emerged by our System Biology approach. In conclusion, our findings suggest that in vivo cell ageing and in vitro subculturing must be taken into account when testing regenerative tissue strategies that use progenitor cells. Indeed, cells (e.g. MSCs and PDPCs) from the earliest subculture passages could be useful to validate any bone tissue engineering strategies, whilst the later ones could be used to test in vitro scaffolds for regenerative medicine approaches in elderly.This work was supported by grants from MIUR (Project PRIN 2010, MIND-2010J8RYS7)

Aging of periosteal-derived stem cells during expansion: an alternative tool for a customized bone regenerative strategy

Increased in life expectancy points out the necessity for tailored strategies to restore bone loss due to trauma and/or disease in elderly. Moreover, there is a compelling need for improved cell systems to test scaffolds interfacing with an “aging” tissue. For skeletal tissue regeneration, periosteal-derived stem cells (PDPCs) could represent an easily recruited source of Mesenchymal stromal cells (MSCs) [1,2]. This study investigated the effects of long-term in vitro expansion on the stability and function of PDPCs, since extensive culture expansion is usually performed to obtain clinically relevant cell numbers, but its impact on cell behaviour is still unclear. An integrated approach based on flow cytometry, ultrastructural and quantitative Real time PCR (qRT-PCR) analyses was adopted. Senescent cell data were compared with those of cells isolated from differently aged subjects. Both replicative-senescent PDPCs and cells isolated from old donors were permanently blocked in G1 phase of cell cycle, through a pathway that seemed to involve nitric oxide (NO) production and the expression of tumour suppressor proteins p16 or p53, respectively. Changes in the expression of MSC surface markers were detected in PDPCs during subculturing, whilst it was superimposable in young and aged PDPCs. Cytofluorimetric analysis of the physical parameters (i.e. FSC and SSC) showed a trend toward an increase in cell dimension and internal complexity in both populations analysed. This data was consistent with morphological observation that also evidenced similar alterations in mitochondrial shape. In addition, an intense autophagic activity in early passage PDPCs was observed, whilst in the late passages cells had a robust protein synthesis activity that could be related with “senescence-associated secretory phenotype” (SASP). In conclusion, the morphofunctional similarities detected in replicative-senescent and aged PDPCs suggest that their long-term expansion could be a reproducible and useful tool to mimic in vivo ageing

Age-related changes in human periosteal derived stem cells: a matter for effective bone regeneration strategies

Possible age-related changes in Mesenchymal Stem Cells (MSCs) are of great inter- est in view of their use for regenerative medicine approaches also in the elderly. Con- sidering the primary role of periosteum in bone biology and to acquire data for a cell- based therapy stimulating graft osseointegration, we tried to identify specific aging markers or pattern of expression in human periosteal precursor cells (PDPCs). To this aim periosteal tissue was obtained from differently aged healthy subjects, gender matched with a mean age of 16, 28, 63 and 92 years. Immunohistochemical detection of Ki67 and p53, Nitric Oxide (NO) production and qRT- PCR of a selected gene pan- el for early osteoblastic differentiation (bmp2 and runx2) and bone remodelling (IL-6, RANKL and OPG) were evaluated. Our data evidenced that both Ki67 and p53 rep- resent striking markers of cell-cycle arrest in these cells and that their expression cor- relates with NO production. In addition, age affects genes involved in bone remodel- ling, with a significant increase in IL-6 mRNA expression as well as in RANKL/OPG ratio. As far as NO release is concerned, our data showed higher levels in PDPCs iso- lated from the elderly and a good correlation with the immunohistochemical analysis Moreover, mathematical modelling, in silico simulations and biochemical experiments were combined to investigate about possible underlying quantitative correlations. A clear one-term exponential relationship emerged from a comparison of involved marker trends against age of donors concerning measured NO concentration / Ki67 ratio. This analytical approach confirmed Ki67 as a senescence marker to be focused on. We believe that this study, taking into account age-related changes in human PDPCs, opens up new regenerative medicine strategies for aged bone and/or bone metabolic diseases