Osteo-regenerative potential of ovarian granulosa cells: an in vitro and in vivo study.

Granulosa cells (GC) express stemness markers and can differentiate into cell types not present within the follicles. Given that follicles at different stages of development populate the ovary, we undertook this research in the pig model to identify the stage of follicle, growing or luteinizing, from which GC with the best regenerative potential can be retrieved. Growing follicles were isolated from prepubertal gilts 50 h after equine chorionic gonadotropin (eCG) (1,200 IU) administration. Luteinizing follicles were obtained from prepubertal gilts treated with eCG (1,200 IU) followed, 60 h later, by hCG (500 IU). The follicles were isolated 30 h after hCG. The GC isolated from growing (GGC) and from luteinizing (LGC) follicles were expanded in vitro for three passages and exposed to osteogenic medium to trigger differentiation. The GC incorporated in PLGA scaffolds were cultured in osteogenic medium for 2 wks and then implanted subcutaneously in the dorsal region of SCID mice to assess their osteogenic potential in vivo. In addition to the typical granulosa cells characteristics (inhibin, progesterone and estrogen production and FSH receptors), GGC and LGC showed a diffused expression of the stemness markers Sox2, Nanog and TERT immediately after isolation. Expansion caused in both cell types a rapid disappearance of granulosa cell characters while it did not modify stemness marker expression. Osteogenic medium induced a marked extracellular matrix mineralization and alkaline phosphatase activation in LGC, clearly detectable after two wks, while the process was much lighter in GGC, where it became evident after 3 wks. Osteocalcin and Runx2 expressions were upregulated and stemness markers downregulated by osteogenic medium. The GC loaded implants, retrieved 8 wks after transplantation, had viable GC surrounding the several nodules of calcifications recorded. Similar effects were induced by GGC and LGC while calcification nodules were not recorded when scaffolds without cells were implanted. These data confirm that GC, expanded in vitro undergo progressive de-differentiation retaining their plasticity and demonstrate that both GGC and LGC have osteogenic potential, luteinizing cells being more efficient. Transplanted in SCID mice, GC participate in new bone formation, thus confirming their therapeutic potential.

Experimental study on allografts of amniotic epithelial cells in calcaneal tendon lesions of sheep.

An experimental protocol was designed to study the survival and behaviour of an allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted on three healthy adult sheep. A core lesion was created in both calcaneal tendons under ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml saline. The AECs were obtained from a 60-80-day-old fetus and cultured under standard conditions. After 15 days of collagenase treatment, 2 x 10(6) AECs stained with a vital membrane fluorescent probe (PHK26) were injected under US guidance in 500 microl saline solution into the lesion of one limb. The contralateral untreated limb was used as a control. Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the lesion without any adverse reactions. The damaged tissue of the treated tendons showed a high number of reparative cells in active proliferation that were accumulating collagen within the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized into parallel arrays of fibers oriented along the longitudinal axis of the tendon.

Follicle activation involves vascular endothelial growth factor production and increased blood vessel extension.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.

Vascular endothelial growth factor production in growing pig antral follicles.

Angiogenesis is the process that drives blood vessel development in growing tissues in response to the local production of angiogenic factors. With the present research the authors have studied vascular endothelial growth factor (VEGF) production in ovarian follicles as a potential mechanism of ovarian activity regulation. Prepubertal gilts were treated with 1250 IU equine chorionic gonadotropin (eCG) followed 60 h later by 750 IU of human chorionic gonadotropin (hCG) in order to induce follicle growth and ovulation. Ovaries were collected at different times of the treatment and single follicles were isolated and classified according to their diameter as small (<4 mm), medium (4-5 mm), or large (>5 mm). VEGF levels were measured in follicular fluid by enzyme immunoassay, and VEGF mRNA content was evaluated in isolated theca and granulosa compartments. Equine chorionic gonadotropin stimulated a prompt follicular growth and induced a parallel evident rise in VEGF levels in follicular fluid of medium and large follicles. Analysis of VEGF mRNA levels confirmed the stimulatory effect of eCG, showing that it is confined to granulosa cells, whereas theca cells maintained their VEGF steady state mRNA. Administration of hCG 60 h after eCG caused a dramatic drop in follicular fluid VEGF that reached undetectable levels in 36 h. A parallel reduction in VEGF mRNA expression was recorded in granulosa cells. The stimulating effect of eCG was also confirmed by in vitro experiments, provided that follicles in toto were used, whereas isolated follicle cells did not respond to this hormonal stimulation. Consistent with the observation in vivo, granulosa cells in culture reacted to hCG with a clear block of VEGF production. These results demonstrate that while follicles of untreated animals produce stable and low levels of the angiogenic factor, VEGF markedly rose in medium and large follicles after eCG administration. The increasing levels, essentially attributable to granulosa cells, are likely to be involved in blood vessel development in the wall of growing follicles, and may play a local key role in gonadotropin-induced follicle development. When ovulation approaches, under the effect of hCG, the production of VEGF is switched off, probably creating the safest conditions for the rupture of the follicle wall while theca cells maintained unaltered angiogenic activity, which is probably required for corpus luteum development.

The phosphotransferase activity of cytosolic 5'-nucleotidase; a purine analog phosphorylating enzyme.

Cytosolic 5'-nucleotidase is involved in the phosphorylation of several purine nucleoside analogs,used as antiviral and chemotherapeutic agents. In order to assess its role in the mechanisms of activation and inactivation of purine prodrugs, it is essential to study the regulation of both hydrolase and phosphotransferase activities of the enzyme. Using a zone capillary electrophoresis apparatus, we were able to separate substrates and products of the reactions catalyzed by cytosolic 5'-nucleotidase. The method overcomes the frequent unavailability of radiolabeled substrates, and allows the influence of possible effectors and/or experimental conditions on both enzyme activities to be evaluated simultaneously. Results showed that the enzyme was able to phosphorylate several nucleosides and nucleoside analogs with the following efficiency: inosine and 2'-deoxyinosine > 2',3'-dideoxyinosine > 6-chloropurineriboside > 6-hydroxylaminepurine riboside> 2,6-diaminopurine riboside > adenosine > cytidine > deoxycoformycin > 2'deoxyadenosine. This is the first report of deoxycoformycin phosphorylation catalyzed by a 5'-nucleotidase purified from eukaryotic cells. The optimum pH for nucleoside monophosphate hydrolysis was 6.5, slightly more acidic than the optimum pH for the transfer of the phosphate, which was 7.2. Finally, the presence of a suitable substrate for the phosphotransferase activity of cytosolic 5'-nucleotidase caused a stimulation of the rate of formation of the nucleoside. The results suggest the requirements for phosphorylation of nucleoside analogs are a purine ring and the presence of an electronegative group in the 6 position. The stimulation of the rate of nucleoside monophosphate disappearance exerted by the phosphate acceptor suggests that the hydrolysis of the phosphoenzyme intermediate is the rate-limiting step of the process.

Purine enzyme profile in human colon-carcinoma cell lines and differential sensitivity to deoxycoformycin and 2'-deoxyadenosine in combination.

Different cell lines, 2 from human colon carcinoma (LoVo and HT29) and 1 from Chinese hamster ovary (CHO K-I), were examined to assess the effect of deoxycoformycin (dCF), an inhibitor of adenosine deaminase (ADA), and 2'-deoxyadenosine (dAdo) on their growth. When used alone, neither dCF or dAdo were cytotoxic for the 3 cell lines, while their combination caused inhibition of cell growth, with the following sensitivity: CHO K-I > LoVo > HT29. We studied the pattern of enzymatic activities involved in the metabolism of dAdo in the 3 cell lines. The phosphorylation of dAdo by adenosine kinase appears to play a central role in the toxicity of the deoxynucleoside in combination with dCF. In fact, CHO K-I cells, which are the most sensitive, possess the highest level of this enzyme. Moreover, the cytotoxic effect was almost completely reversed in the 3 cell lines when inhibitors of adenosine kinase, such as 5'-amino-5'-deoxyadenosine and iodotubercidine, were added to the culture medium together with dCF and dAdo. In addition, baby hamster kidney (BHK) adenosine-kinase-deficient (AK-) cells were highly resistant to this treatment. Uptake inhibition of dAdo using dipyridamole also caused reversal of the toxicity. The AMP and deoxyAMP dephosphorylating activities, much lower in the CHO K-I cells, also appear to play a central role in the toxicity of dAdo when adenosine deaminase is inhibited. However, our data suggest that other factors may modulate the toxic effect, such as S-adenosyl-homocysteine-hydrolase inhibition by dAdo at high concentrations.

The bifunctional cytosolic 5'-nucleotidase: regulation of the phosphotransferase and nucleotidase activities.

The cytosolic 5'-nucleotidase specific for IMP, GMP, and their deoxyderivatives has been purified approximately 1000 times from calf thymus. The enzyme, in the presence of a suitable nucleoside, can act as a phosphotransferase, catalyzing the transfer of the phosphate moiety from a nucleoside monophosphate donor to a nucleoside acceptor, thus operating as an interconverting activity. This phosphorylating activity has drawn the attention of several research groups because the cytosolic 5'-nucleotidase represents the only cellular enzyme able to phosphorylate inosine and guanosine analogs, which are not substrates of known cellular nucleoside kinases. In this paper, we report the kinetic parameters of the bifunctional enzyme and its response to variations in adenylate energy charge. The results seem to indicate that in the presence of physiological concentrations of ATP and phosphate, the enzyme behaves mainly as a phosphotransferase, its activity being dependent only on the availability of a suitable nucleoside.

Relationship between the levels of purine salvage pathway enzymes and clinical/biological aggressiveness of human colon carcinoma.

A large series of samples obtained after surgical resection of intestinal mucosa of patients affected by intestinal carcinoma was examined in order to define possible relationships between levels of enzymes involved in the purine salvage pathway and clinical/biological parameters of aggressiveness and invasiveness. The results confirm our previous observation on a different pattern of purine salvage enzymes in tumor as compared to normal colon tissues (Camici et al., 1990). In fact, we observed in human colon tumor tissues a significant enhancement of the three enzymes involved in the synthesis of IMP, hypoxanthine guanine phosphoribosyltransferase (HGPRT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP). On the other hand, no variation was observed in the 5'-nucleotidase and alkaline phosphatase activities. While we could not find a significant correlation between HGPRT, ADA and PNP activities and histologic grading or biological parameters of tumor aggressiveness, the significant correlation with the extent of disease, as expressed by the Dukes' stage, would demonstrate at least for human colon tumors, a relationship between enzyme activity and tumor invasiveness.

Cytosolic 5'-nucleotidase/nucleoside phosphotransferase: a nucleoside analog activating enzyme?

Nucleoside phosphotransferase acting on inosine and deoxyinosine has been partially purified from cultured Chinese hamster lung fibroblasts (V79). The activity is associated with a cytosolic 5'-nucleotidase acting on IMP and deoxyIMP. The transfer of the phosphate group from IMP to inosine catalyzed by this enzyme was activated by ATP and 2,3-bisphosphoglycerate. Inosine, deoxyinosine, guanosine, deoxyguanosine, and the nucleoside analogs 2',3'-dideoxyinosine and 8-azaguanosine are substrates, while adenosine and deoxyadenosine are not. IMP, deoxyIMP, GMP, and deoxyGMP are the best phosphate donors. The cytosolic 5'-nucleotidase/phosphotransferase substrate, 8-azaguanosine, was found to be very toxic for cultured fibroblasts (LD50 = 0.32 microM). Mutants resistant to either 8-azaguanosine and the correspondent base 8-azaguanine were isolated and characterized. Our results indicated that the 8-azaguanosine-resistant cells were lacking both cytosolic 5'-nucleotidase and hypoxanthine-guanine phosphoribosyltransferase, while 8-azaguanine resistant cells were lacking only the latter enzyme. Despite this observation, both mutants displayed 8-azaguanosine resistance, thus indicating that cytosolic 5'-nucleotidase is not essential for the activation of this nucleoside analog.

Membrane-bound 5'-nucleotidase/nucleoside phosphotransferase from Bacillus cereus.

1. A search for nucleoside phosphotransferase activity in Bacillus cereus led to the following results: (i) The phosphotransferase activity was associated with a membrane bound 5'-nucleotidase. (ii) The enzyme phosphorylates both purine and pyrimidine nucleosides as well as 2',3'-dideoxyinosine. (iii) The enzyme was inhibited by adenylic nucleotide di- and triphosphates, and its nucleotidase activity was increased in the presence of inosine as phosphate acceptor. 2. Bacterial and vertebrate 5'-nucleotidases with phosphotransferase activity differ for several characteristics, such as cellular location, substrate specificity, magnesium requirement and regulation.