In vitro cultured progenitors and precursors of cardiac cell lineages from human normal and post-ischemic hearts.

The demonstration of the presence of dividing primitive cells in damaged hearts has sparked increased interest about myocardium regenerative processes. We examined the rate and the differentiation of in vitro cultured resident cardiac primitive cells obtained from pathological and normal human hearts in order to evaluate the activation of progenitors and precursors of cardiac cell lineages in post-ischemic human hearts. The precursors and progenitors of cardiomyocyte, smooth muscle and endothelial lineage were identified by immunocytochemistry and the expression of characteristic markers was studied by western blot and RT-PCR.The amount of proteins characteristic for cardiac cells (alpha-SA and MHC, VEGFR-2 and FVIII, SMA for the precursors of cardiomyocytes, endothelial and smooth muscle cells, respectively) inclines toward an increase in both alpha-SA and MHC. The increased levels of FVIII and VEGFR2 are statistically significant, suggesting an important re-activation of neoangiogenesis. At the same time, the augmented expression of mRNA for Nkx 2.5, the trascriptional factor for cardiomyocyte differentiation, confirms the persistence of differentiative processes in terminally injured hearts. Our study would appear to confirm the activation of human heart regeneration potential in pathological conditions and the ability of its primitive cells to maintain their proliferative capability in vitro. The cardiac cell isolation method we used could be useful in the future for studying modifications to the microenvironment that positively influence cardiac primitive cell differentiation or inhibit, or retard, the pathological remodeling and functional degradation of the heart

Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts

Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed

Effect Of Priming On Seed Vigor Of Wheat (Triticum Aestivum L.)

Priming is a process that controls the process of hydration of seeds for the ongoing metabolic processes before germination. Research on priming was conducted at ICERI seed laboratory from May to September 2009 to evaluate the effect of different priming methods on wheat seed vigor. Physical properties and chemical composition of seed were evaluated before seeds were treated. The priming treatment were conducted by soaking 250 g of seed in 500 mL of solution for hydropriming and halopriming. Two seed lots of Nias and Dewata variety were subjected to heated and unheated distilled water for 12hours and subjected to KCl and CaCl2 at 10, 20, and 30 ppm and unprimed seed. The experiment were arranged in completely randomized design, replicated thrice. Vigor evaluation by observed seed germination, simultaneity growth, germination rate, seedling dry weight, electric conductivity of seed leakage and length of primary root. The results showed that highest germination, simultaneity growth, seedling dry weight, and length of primary root, were priming treatment with KCl 30 ppm and CaCl2 20 and 30 ppm. Priming with distilled water for 12 hours gave higher germination percentage and simultaneity growth

Thyroid Cancer and Fibroblasts

Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs

Analysis of SOD3 and Akt in ascending aortic aneurysm

Ascending aortic aneurysm (AsAA) is divided into three different forms: syndromic, familial non-syndromic, and degenerative. Bicuspid aortic valve (BAV), occuring in 2% of the population, is the most frequent cardiac congenital abnormality, associated to AsAA. All the different forms of AsAAs are a consequence of cystic medial necrosis (CMN), characterized by apoptotic loss of smooth muscle cells (SMCs), fragmentation of elastic and collagen fibers and increased accumulation of mucoid material. The extracellular superoxide dismutase (SOD3) is a Cu/Zn enzyme, affecting redox state and homeostasis of extracellular matrix (ECM) (1). Moreover, the outsidein signalling from ECM modulates intracellular pathways regulating many cellular functions. The multifunctional Akt pathway affects survival and cellular proliferation and has important effects on the cardiovascular function. In this study we examined the relevance of SOD3 and Akt in AsAA pathogenesis. To this aim, the SOD3 and Akt protein levels were evaluated in normal ascending aortic tissues (n=6) and in tissues from AsAAs associated both to tricuspid aortic valve (TAV) (n=6) and BAV (n=6); moreover, we measured SOD3 activity in sera from healthy donors and patients with AsAA. Our data showed a reduction of SOD3 and phospho-Akt (pAkt) protein levels in AsAAs from BAV patients compared to normal donors; on the other hand, no differences emerged in SOD3 activity. Furthermore, immunohistochemical analysis performed on normal and pathological ascending aortic tissues showed a SOD3 immunostaining in both extracellular space and tunica media cells from normal ascending aortic tissues; conversely, no SOD3 immunostaining was detected in AsAAs tissues from both TAV and BAV patients. Our data show that SOD3 and pAkt could be associated to AsAA pathogenesis and suggest a link between ECM homeostasis and Akt survival pathway

Generation of spheroids from human primary myofibroblasts: an experimental system to study myofibroblasts deactivation

Fibroblasts represent a heterogeneous cell population, that in adult body maintains the homeostasis of the extracellular matrix (ECM) and can acquire an immunoregulatory phenotype. Indeed, activated fibroblasts produce large amounts of cyclooxygenase-2 (COX-2) and proinflammatory cytokines (1). The activation of fibroblasts is represented by their differentiation into myofibroblasts. This process, either in wound healing or cancer tissue, is associated with the expression of alpha-smooth muscle actin (alpha-SMA), increased levels of growth factors and ECM-degrading proteases (2). Moreover, myofibroblasts form clusters in wound healing process and hypertrophic scars. In particular, cell clusters of hypertrophic scars are represented by nodules of myofibroblasts (3). It is known that human dermal fibroblasts established from neonatal foreskin, and forced in vitro to form clusters named spheroids, are activated to produce massive amounts of COX-2, prostaglandins and proinflammatory cytokines: this process leads to a programmed necrosis, designated “nemosis” (1). In the present study we generated spheroids from human primary myofibroblasts of skin, to evaluate necrotic, inflammation and activation markers during myofibroblasts clustering. Western blotting analysis, showing low levels of COX-2 and a significant decrease of alpha-SMA in protein extracts of spheroids, led to hypothesize that myofibroblasts have undergone a deactivation process within spheroids. This hypothesis is confirmed by cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines, by confocal immunofluorescence analysis of connexin 43 and immunohistochemical evaluation of proliferation marker Ki-67. This work could represent an experimental model to study myofibroblasts deactivation and highlights an alternative process regulating the turnover of myofibroblasts

Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation

Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells

Insights into Melanoma Fibroblast Populations and Therapeutic Strategy Perspectives: Friends or Foes?

: Cutaneous melanoma (CM) is an aggressive and highly metastatic solid tumor associated with drug resistance. Before 2011, despite therapies based on cytokines or molecules inhibiting DNA synthesis, metastatic melanoma led to patient death within 18 months from diagnosis. However, recent studies on bidirectional interactions between melanoma cells and tumor microenvironment (TME) have had a significant impact on the development of new therapeutic strategies represented by targeted therapy and immunotherapy. In particular, the heterogeneous stromal fibroblast populations, including fibroblasts, fibroblast aggregates, myofibroblasts, and melanoma associated fibroblasts (MAFs), represent the most abundant cell population of TME and regulate cancer growth differently. Therefore, in this perspective article, we have highlighted the different impacts of fibroblast populations on cancer development and growth. In particular, we focused on the role of MAFs in sustaining melanoma cell survival, proliferation, migration and invasion, drug resistance, and immunoregulation. The important role of constitutively activated MAFs in promoting CM growth and immunoediting makes this cell type a promising target for cancer therapy

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes.

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 isclearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distributionof the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region