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PubMed2000

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis.

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.

Animals↗
PubMed1997

Development of inflammatory angiogenesis by local stimulation of Fas in vivo.

Fas-Fas ligand interaction is thought to be a crucial mechanism in controlling lymphocyte expansion by inducing lymphocyte apoptosis. However, Fas is also broadly expressed on nonlymphoid cells, where its function in vivo remains to be determined. In this study, we describe the development of inflammatory angiogenesis induced by agonistic anti-Fas mAb Jo2 in a murine model where Matrigel is used as a vehicle for the delivery of mediators. The subcutaneous implants in mice of Matrigel containing mAb Jo2 became rapidly infiltrated by endothelial cells and by scattered monocytes and macrophages. After formation and canalization of new vessels, marked intravascular accumulation and extravasation of neutrophils were observed. Several mast cells were also detected in the inflammatory infiltrate. The phenomenon was dose and time dependent and required the presence of heparin. The dependency on activation of Fas is suggested by the observation that the inflammatory angiogenesis was restricted to the agonistic anti-Fas mAb and it was absent in lpr Fas-mutant mice. Apoptotic cells were not detectable at any time inside the implant or in the surrounding tissue, suggesting that angiogenesis and cell infiltration did not result from recruitment of phagocytes by apoptotic cells but rather by a stimulatory signal through Fas-engagement. These findings suggest a role for Fas-Fas ligand interaction in promoting local angiogenesis and inflammation.

Animals↗
PubMed2002

Systemic delivery of human growth hormone using genetically modified tissue-engineered microvascular networks: prolonged delivery and endothelial survival with inclusion of nonendothelial cells.

Endothelial cells have the potential to provide efficient long-term delivery of therapeutic proteins to the circulation if a sufficient number of genetically modified endothelial cells can be incorporated into the host vasculature and if these cells persist for an adequate period of time. Here we describe the ability of nonendothelial cells to modulate the survival of implanted endothelial cells and their incorporation into host vasculature. Bovine aortic endothelial cells (BAECs) suspended in Matrigel and cultured in vitro remained spherical and decreased in number over time. Subcutaneous implantation of gels containing BAECs secreting human growth hormone (hGH) in mice initially resulted in detectable plasma hGH levels, which were undetectable after 2 weeks. When mixed with fibroblasts and suspended in Matrigel, hGH-secreting BAECs formed microvascular networks in vitro. Implantation of these gels resulted in plasma hGH levels that decreased slightly over 2 weeks and then remained stable for at least 6 weeks. BAECs incorporated into blood vessels within both the implant and fibrous capsule that surrounded and invaded implants. Within implants containing BAECs and fibroblasts, viable BAECs were present for at least 6 weeks at a higher density than in implants containing BAECs alone at 3 weeks. These results indicate that implanted BAECs can incorporate into host blood vessels and that inclusion of fibroblasts in this system prolongs BAEC survival and hGH delivery.

Animals↗
PubMed2004

Pomegranate extracts potently suppress proliferation, xenograft growth, and invasion of human prostate cancer cells.

We completed a multicenter study of the effects of pomegranate cold-pressed (Oil) or supercritical CO(2)-extracted (S) seed oil, fermented juice polyphenols (W), and pericarp polyphenols (P) on human prostate cancer cell xenograft growth in vivo, and/or proliferation, cell cycle distribution, apoptosis, gene expression, and invasion across Matrigel, in vitro. Oil, W, and P each acutely inhibited in vitro proliferation of LNCaP, PC-3, and DU 145 human cancer cell lines. The dose of P required to inhibit cell proliferation of the prostate cancer cell line LNCaP by 50% (ED(50)) was 70 microg/mL, whereas normal prostate epithelial cells (hPrEC) were significantly less affected (ED(50) = 250 g/mL). These effects were mediated by changes in both cell cycle distribution and induction of apoptosis. For example, the androgen-independent cell line DU 145 showed a significant increase from 11% to 22% in G(2)/M cells (P <.05) by treatment with Oil (35 microg/mL) with a modest induction of apoptosis. In other cell lines/treatments, the apoptotic response predominated, for example, in PC-3 cells treated with P, at least partially through a caspase 3-mediated pathway. These cellular effects coincided with rapid changes in mRNA levels of gene targets. Thus, 4-hour treatment of DU 145 cells with Oil (35 microg/mL) resulted in significant 2.3 +/- 0.001-fold (mean +/- SEM) up-regulation of the cyclin-dependent kinase inhibitor p21((waf1/cip1)) (P <.01) and 0.6 +/- 0.14-fold down-regulation of c-myc (P <.05). In parallel, all agents potently suppressed PC-3 invasion through Matrigel, and furthermore P and S demonstrated potent inhibition of PC-3 xenograft growth in athymic mice. Overall, this study demonstrates significant antitumor activity of pomegranate-derived materials against human prostate cancer.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Inhibition of human collagenase activity by antisense oligonucleoside methylphosphonates.

Oligodeoxyribonucleoside methylphosphonates (d-OMP) were synthesized whose sequences are complementary to sequences found in the mRNA coding for the 72-kDa (MMP-2) or 92-kDA (MMP-9) forms of human collagenase i.v., matrix metalloproteinases (MMP) whose excessive secretion correlates with the metastatic potential of tumor cells. The effects of these oligomers on MMP-2 and MMP-9 activities secreted by HT1080 cells, a human fibrosarcoma cell line, were studied using a gelatin zymography assay. A d-OMP, M2.3, complementary to nucleotides 14 to 28 of the initiation codon region of MMP-2 mRNA selectively inhibited MMP-2 activity, whereas a d-OMP, M9.1, which was targeted to nucleotides -19 to -5 of the 5'-untranslated region of MMP-9 mRNA selectively inhibited MMP-9 activity over the concentration range 5-50 microM. At 100 microM concentration, both M2.3 and M9.1 inhibited the activities of both MMP-2 and MMP-9. These oligomers were completely stable under cell culture conditions and did not appear to adversely affect cell growth after 48 hours at concentrations up to 100 microM, although 100 microM M9.1 did reduce cell growth 30% after prolonged, 120-hours exposure. Other d-OMP tested either had no effect on collagenase activity or inhibited both MMP-2 and MMP-9 activities. The latter oligomer was complementary to MMP-2 mRNA and partially complementary to MMP-9 mRNA. Oligomer M2.3 was also tested for its effects on the morphology of malignant human lung cells, BZR-T33, growing on the surface of reconstituted base membrane, Matrigel, in culture. In absence of oligomer, the BZR-T33 cells formed extensive networks indicative of the ability of the cells to invade the Matrigel substrate. In the presence of 100 microM M2.3, BZR-T33 formed colonies of rounded cells, a morphology typical of noninvasive cells. Other non-complementary d-OMP had no effect on the morphology of BZR-T33 under these conditions. These results suggest that antisense d-OMP may be useful for inhibiting expression of collagenase in human tumor cells and for studying the role of collagenase expression in tumor cell metastasis.

Collagenases↗
PubMed2006

Engineering three-dimensional pulmonary tissue constructs.

In this paper, we report on engineering 3-D pulmonary tissue constructs in vitro. Primary isolates of murine embryonic day 18 fetal pulmonary cells (FPC) were comprised of a mixed population of epithelial, mesenchymal, and endothelial cells as assessed by immunohistochemistry and RT-PCR of 2-D cultures. The alveolar type II (AE2) cell phenotype in 2-D and 3-D cultures was confirmed by detection of SpC gene expression and presence of the gene product prosurfactant protein C. Three-dimensional constructs of FPC were generated utilizing Matrigel hydrogel and synthetic polymer scaffolds of poly-lactic-co-glycolic acid (PLGA) and poly-L-lactic-acid (PLLA) fabricated into porous foams and nanofibrous matrices, respectively. Three-dimensional Matrigel constructs contained alveolar forming units (AFU) comprised of cells displaying AE2 cellular ultrastructure while expressing the SpC gene and gene product. The addition of tissue-specific growth factors induced formation of branching, sacculated epithelial structures reminiscent of the distal lung architecture. Importantly, 3-D culture was necessary for inducing expression of the morphogenesis-associated distal epithelial gene fibroblast growth factor receptor 2 (FGFr2). PLGA foams and PLLA nanofiber scaffolds facilitated ingrowth of FPC, as evidenced by histology. However, these matrices did not support the survival of distal lung epithelial cells, despite the presence of tissue-specific growth factors. Our results may provide the first step on the long road toward engineering distal pulmonary tissue for augmenting and/or replacing dysfunctional native lung in diseases, such as neonatal pulmonary hypoplasia.

Animals↗
PubMed2002

Function of the tetraspanin CD151-alpha6beta1 integrin complex during cellular morphogenesis.

Upon plating on basement membrane Matrigel, NIH3T3 cells formed an anastomosing network of cord-like structures, inhibitable by anti-alpha6beta1 integrin antibodies. For NIH3T3 cells transfected with human CD151 protein, the formation of a cord-like network was also inhibitable by anti-CD151 antibodies. Furthermore, CD151 and alpha6beta1 were physically associated within NIH3T3 cells. On removal of the short 8-amino acid C-terminal CD151 tail (by deletion or exchange), exogenous CD151 exerted a dominant negative effect, as it almost completely suppressed alpha6beta1-dependent cell network formation and NIH3T3 cell spreading on laminin-1 (an alpha6beta1 ligand). Importantly, mutant CD151 retained alpha6beta1 association and did not alter alpha6beta1-mediated cell adhesion to Matrigel. In conclusion, the CD151-alpha6beta1 integrin complex acts as a functional unit that markedly influences cellular morphogenesis, with the CD151 tail being of particular importance in determining the "outside-in" functions of alpha6beta1-integrin that follow ligand engagement. Also, antibodies to alpha6beta1 and CD151 inhibited formation of endothelial cell cord-like networks, thus pointing to possible relevance of CD151-alpha6beta1 complexes during angiogenesis.

3T3 Cells↗
PubMed1997

Thrombospondin-1, a natural inhibitor of angiogenesis, regulates platelet-endothelial cell adhesion molecule-1 expression and endothelial cell morphogenesis.

Expression of thrombospondin-1 (TS1) in polyoma middle-sized T (tumor)-transformed mouse brain endothelial cells (bEND.3) restores a normal phenotype and suppresses their ability to form hemangiomas in mice. We show that TS1 expression results in complete suppression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) expression and altered cell-cell interactions in bEND.3 cells. To further investigate the role of PECAM-1 in regulation of endothelial cell-cell interactions and morphogenesis, we expressed human (full length) or murine (delta 15) PECAM-1 isoforms in TS1-transfected bEND.3 (bEND/TS) cells. Expression of either human or murine PECAM-1 resulted in an enhanced ability to organize and form networks of cords on Matrigel, an effect that was specifically blocked by antibodies to PECAM-1. Anti-PECAM-1 antibodies also inhibited tube formation in Matrigel by normal human umbilical vein endothelial cells. However, PECAM-1-transfected bEND/TS cells did not regain the ability to form hemangiomas in mice and the expressed PECAM-1, unlike the endogenous PECAM-1 expressed in bEND.3 cells, failed to localize to sites of cell-cell contact. This may be, in part, attributed to the different isoforms of PECAM-1 expressed in bEND.3 cells. Using reverse transcription-polymerase chain reaction, we determined that bEND.3 cells express mRNA encoding six different PECAM-1 isoforms, the isoform lacking both exons 14 and 15 (delta 14&15) being most abundant. Expression of the murine delta 14&15 PECAM-1 isoform in bEND/TS cells resulted in a similar phenotype to that described for the full-length human or murine delta 15 PECAM-1 isoform. The delta 14&15 isoform, despite the lack of exon 14, failed to localize to sites of cell-cell contact even in clones that expressed it at very high levels. Thus, contrary to recent reports, lack of exon 14 is not sufficient to result in junctional localization of PECAM-1 isoforms in bEND/TS cells.

Animals↗
PubMed1997

Clonal heterogeneity of the growth and invasive response of a human breast carcinoma cell line to parathyroid hormone-related peptide fragments.

It has been previously reported that 8701-BC cells, derived from a primary carcinoma of the breast, constitutively express parathyroid hormone (PTH)-related peptide (PTHrP) and PTH/PTHrP receptor (PTH/PTHrP-R) genes, that N-terminal, mid-regional and C-terminal immunoreactive PTHrP can be found in cell conditioned medium and, furthermore, that exogenously added PTHrP (1-34), (67-86) and, to a minor extent, (107-139) are anti-mitogenic but promote Matrigel invasion by this cell line. It has also been reported that PTHrP gene expression is selectively switched on in those 8701-BC clonal lines endowed with a higher proliferation rate and invasive ability in vitro. Here we have first examined the presence of PTH/PTHrP-R transcript in the different 8701-BC clones by PCR and Southern blot analysis. Second, we have studied the growth and invasive response in vitro to PTHrP fragments by some of these clones, i.e. BC-3A, BC-61 and BC-66, selected on the basis of their lower (BC-3A) or higher (BC-1 and BC-66) Matrigel invasion ability and their expression of PTHrP (positive for BC-61 and BC-66) and PTH/PTHrP-R (positive for BC-61). Our data show the existence of clonal heterogeneity for PTH/PTHrP-R mRNA and for the proliferative and invasive responses elicited by treatment with diverse PTHrP fragments. In particular: (i) the sensitivity to PTHrP (1-34) is restricted due to the uneven expression of PTH/PTHrP-R; (ii) BC-3A cells (the less 'aggressive' clone) are resistant to the anti-mitogenic effect of the PTHrP domains and, most noticeably, exhibit a growth-potentiating response to PTHrP (67-86) opposite to that found for both the parental 8701-BC cells and the two other clones; (iii) all PTHrP fragments tested induced the expression of a growth-restraining and invasion-promoting phenotype by BC-61 cells (one of the more 'aggressive' clones). Present data in vitro support the hypothesis that in vivo PTHrP may be a key element in local control of the invasive process during breast carcinoma development and that its role may be, in turn, dependent upon the biological characteristics and the level of malignancy of the target cells within the multiclonal population of a primary tumour.

Breast Neoplasms↗
PubMed1997

Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells.

Invasive prostatic carcinomas and prostatic intraepithelial neoplasia (PIN) are characterized by a loss of normal cell organization, cell polarity, and cell:cell and cell:basement membrane adhesion. The objective of this study was to establish in vitro three-dimensional (3-D) cell models which can be used to investigate mechanisms involved in acinar morphogenesis and differentiation in normal prostatic epithelium and their abnormalities in cancer cells. The process of acinar morphogenesis, including structural and functional differentiation, was investigated by culture on basement membrane gels (Matrigel). The human papillomavirus 18 immortalized, non-tumorigenic cell line RWPE-1, the v-Ki-ras transformed, tumorigenic RWPE-2 cell line derived from RWPE-1 cells (see previous paper pp. 1221-1229) and the human prostatic carcinoma cell line DU-145 were used. When cultured on Matrigel, RWPE-2 cells remain as single cells or form small aggregates and DU-145 cells form large amorphous cell aggregates without any organization or lumen. In contrast, RWPE-1 cells form acini of polarized epithelium with a distinct lumen, show a distinct laminin basement membrane, and express alpha6beta1 integrins at their basal end. Exposure to conditioned medium from NIH 3T3 cultures accelerates glandular morphogenesis. Parallel cultures maintained as monolayers on plastic remain as monolayers. In the presence of the synthetic androgen mibolerone, acinar cells express prostate specific antigen (PSA) as determined by immunostaining. We conclude that normal prostate cells can undergo acinar morphogenesis while tumorigenic cells have lost this ability. The 3-D cultures provide physiologically relevant in vitro models for elucidating regulation of growth, morphogenesis and differentiation in the normal human prostate, for defining heterotypic interactions in benign prostatic hyperplasia and for establishing the basis for the loss of normal cell organization in early neoplastic lesions such as PIN as well as during tumor progression in prostate cancer.

3T3 Cells↗
PubMed2004

Anti-angiogenic activity of inositol hexaphosphate (IP6).

A significant anticancer activity of the naturally occurring carbohydrate inositol hexaphosphate (IP(6)) has been reported against numerous cancer models. Since tumors require angiogenesis for growth and metastasis, we hypothesize that IP(6) reduces tumor growth by inhibiting angiogenesis. Because angiogenesis depends on the interaction between endothelial and tumor cells, we investigated the effect of IP(6) on both. IP(6) inhibited the proliferation and induced the differentiation of endothelial cells in vitro; the growth of bovine aortic endothelial cells (BAECs) evaluated by MTT proliferation assay was inhibited in a dose-dependent manner (IC(50) = 0.74 mM). The combination of IP(6) and vasostatin, a calreticulin fragment with anti-angiogenic activity, was synergistically superior in growth inhibition than either compound. IP(6) inhibited human umbilical vein endothelial cell (HUVEC) tube formation (in vitro capillary differentiation) on a reconstituted extracellular matrix, Matrigel, and disrupted pre-formed tubes. IP(6) significantly reduced basic fibroblast growth factor (bFGF)-induced vessel formation (P < 0.01) in vivo in Matrigel plug assay. Exposure of HepG2, a human hepatoma cell line, to IP(6) for 8 h, resulted in a dose-dependent decrease in the mRNA levels of vascular endothelial growth factor (VEGF), as assessed by RT-PCR. IP(6) treatment of HepG2 cells for 24 h also significantly reduced the VEGF protein levels in conditioned medium, in a concentration-dependent manner (P = 0.012). Thus, IP(6) has an inhibitory effect on induced angiogenesis.

Angiogenesis Inhibitors↗
PubMed2005

Delphinidin, a dietary anthocyanidin, inhibits vascular endothelial growth factor receptor-2 phosphorylation.

Epidemiological studies have shown that a diet rich in fruits and vegetables has a beneficial preventive effect on cardiovascular diseases and cancer by mechanisms that have not yet been elucidated. In this work, we investigated the antiangiogenic activities of anthocyanidins, a class of polyphenols present at high levels in fruits. Among the tested anthocyanidins (cyanidin, delphinidin, malvidin, pelargonidin, peonidin and petunidin), delphinidin was the most potent angiogenic inhibitor. In vitro, low concentrations of delphinidin inhibited vascular endothelial growth factor (VEGF)-induced tyrosine phosphorylation of VEGF receptor (VEGFR)-2, leading to the inhibition of downstream signaling triggered by VEGFR-2. Inhibition of VEGFR-2 by delphinidin inhibited the VEGF-induced activation of ERK-1/2 signaling and the chemotactic motility of human EC as well as their differentiation into capillary-like tubular structures in Matrigel and within fibrin gels. In vivo, delphinidin was able to suppress basic fibroblast growth factor-induced vessel formation in the mouse Matrigel plug assay. The identification of delphinidin as a naturally occurring inhibitor of VEGF receptors suggests that this molecule possesses important antiangiogenic properties that may be helpful for the prevention and treatment of cancer.

Angiogenesis Inhibitors↗
PubMed2006

In vivo inhibition of angiogenesis by a soluble form of melanotransferrin.

Melanotransferrin (MTf), the membrane-bound human melanoma antigen p97, binds to plasminogen and stimulates its activation, thus regulating a crucial step involved in angiogenesis. In our study, a truncated soluble form of MTf (sMTf) inhibits, in a dose-dependent manner, the in vitro tubulogenesis of human umbilical vessel endothelial cells (HUVEC) induced by media conditioned with MTf-expressing cells. Following these results, we used the in vivo Matrigel plug angiogenesis assay to investigate whether truncated sMTf could inhibit neovascularization in mice. We found that basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and MTf-expressing cells strongly stimulate in vivo Matrigel neovascularization. However, subcutaneous (s.c.) administration of truncated sMTf inhibits both bFGF- and VEGF- as well as human melanoma SK-Mel-28-induced angiogenesis. This inhibition was dependent on the truncated sMTf concentration and reached maximal inhibition at 40 mg/kg/week. Furthermore, we found that a single s.c. injection of truncated sMTf into nude mice at 5 mg/kg produced a maximal blood concentration of approximately 40 nM, which is comparable with the level required to inhibit in vitro HUVEC tubulogenesis. Overall, our results strongly suggest that s.c. administration of truncated sMTf may provide a novel therapeutic strategy for the treatment of angiogenesis-related disorders.

Angiogenesis Inhibitors↗
PubMed2002

FRNK blocks v-Src-stimulated invasion and experimental metastases without effects on cell motility or growth.

Focal adhesion kinase (FAK) was first identified as a viral Src (v-Src) substrate, but the role of FAK in Src transformation events remains undefined. We show that stable expression of the FAK C-terminal domain (termed FRNK) in v-Src-transformed NIH 3T3 fibroblasts inhibited cell invasion through Matrigel and blocked experimental metastases in nude mice without effects on cell motility. FRNK inhibitory activity was dependent upon its focal contact localization. FRNK expression disrupted the formation of a v-Src-FAK signaling complex, inhibited p130Cas tyrosine phosphorylation, and attenuated v-Src-stimulated ERK and JNK kinase activation. However, FRNK did not affect v-Src-stimulated Akt activation, cell growth in soft agar, or subcutaneous tumor formation in nude mice. FRNK-expressing cells exhibited decreased matrix metalloproteinase-2 (MMP-2) mRNA levels and MMP-2 secretion. Transient FRNK expression in human 293 cells inhibited exogenous MMP-2 promoter activity and overexpression of wild-type but not catalytically-inactive (Ala-404) MMP-2 rescued v-Src-stimulated Matrigel invasion in the presence of FRNK. Our findings show the importance of FAK in Src-stimulated cell invasion and support a role for Src-FAK signaling associated with elevated tumor cell metastases.

3T3 Cells↗
PubMed2001

Aging lowers steady-state antioxidant enzyme and stress protein expression in primary hepatocytes.

It has been reported that the isolation and culture of primary hepatocytes can compromise cellular ability to constituitively express antioxidant enzyme (AE) genes, making it difficult to study their regulation ex vivo. In the present study, the steady-state expression of manganese-containing superoxide dismutase, copper- and zinc-containing superoxide dismutase, catalase, and glutathione peroxidase was assessed in primary hepatocytes isolated from young and senescent rats and cultured in MATRIGEL: There was no change in steady-state superoxide dismutase protein or activity levels in cells collected from young animals and cultured for 7 days. Catalase expression was initially increased, and then it declined 30%. In contrast, superoxide dismutase expression declined 60% and catalase expression declined 50% in cells from senescent animals. Constitutive and inducible 70-kDa heat shock protein expression increased coincident with declining AE levels in the young cells but not senescent cells. For both age groups, electron micrographs showed rounded hepatocytes with abundant rough endoplasmic reticulum, mitochondria, and peroxisomes. Hepatocytes were organized into clusters of 6-12 cells surrounding a large central lumen devoid of microvilli. Each cluster also contained smaller microvilli-lined lumens between adjacent hepatocytes that resembled canniculi. The plasma membranes of these lumens were sealed from the extracellular space by junctional complexes. Gap junctions in the plasma membrane suggest that hepatocytes were capable of intercellular communication. We conclude that the Matrigel system can be used to study AE regulation in primary hepatocytes from young and senescent animals, provided that experiments can be conducted within a time frame of 5-7 days in culture. These data also support the hypothesis that aging compromises hepatocellular ability to maintain AE status and upregulate stress protein expression.

Aging↗
PubMed1998

Heparin-binding epidermal growth factor significantly improves human blastocyst development and hatching in serum-free medium.

The purpose of this study was to determine the effect of heparin-binding epidermal growth factor (HB-EGF) on human embryo development in vitro from days 2 to 14 post-insemination. Embryos were cultured in a complex serum-free medium (CSFM3) in the absence and presence of 1 nM and 100 nM HB-EGF. Development to the blastocyst stage of A-C-grade embryos (A grade = highest quality) was improved in the presence of 1 nM HB-EGF from 40.7% to 65.4% and significantly increased to 71.0% in the presence of 100 nM HB-EGF (P < 0.05). Moreover, the percentage of blastocysts hatching was improved in the presence of 1 nM HB-EGF from 45.5% to 70.5% and almost doubled to 81.8% (P < 0.05) in the presence of 100 nM HB-EGF. HB-EGF promoted the development of high-grade (classed as BG1) and medium-grade (BG2) blastocysts. There was no difference in blastocyst quality between the control and HB-EGF-treated embryos as assessed by blastocyst cell number and consumption of the major energy substrates, pyruvate and glucose, measured on day 6 of culture. Further development was assessed by culturing the blastocysts on growth factor-reduced Matrigel (GFR-Matrigel). Adherence and outgrowth were observed, with these embryos producing significantly more human chorionic gonadotrophin over days 7-14 compared with those cultured on plastic (47.8 +/- 8.0 mU versus 23.0 +/- 8.6 mU). The addition of recombinant human growth factors to clinical in-vitro fertilization medium may be useful in promoting embryo development with a view to carrying out blastocyst transfers.

Blastomeres↗
PubMed1999

Morphology and functional characteristics of human ovarian microvascular endothelium.

Corpus luteum formation is characterized by a period of extensive vascularization, as capillaries in the thecal layer of the collapsed follicle following ovulation invade the previously avascular granulosa layer. In order to study these processes in vitro we have developed an endothelial cell preparation from the specific microvasculature of the ovarian follicle. Follicular aspirates, obtained at oocyte collection for in-vitro fertilization (IVF), were filtered to obtain fragments of follicle wall. These were set in Matrigel and then cultured allowing the growth of capillary-like structures through the matrix. Upon emergence from the Matrigel the growing cells formed monolayers with the characteristic cobble-stone morphology of endothelial cells. Immunocytochemistry demonstrated the presence of a range of endothelial-specific markers including von Willebrand factor (vWF), Ulex europeus agglutinin (UEA)-1, CD31 and E-selectin, as well as VCAM-1, which is normally associated with stimulated endothelial cells. RT-PCR analysis showed the expression of two receptors for vascular endothelial growth factor (flt-1 and KDR), and the endothelial nitric oxide synthase, adding further evidence of their identity as human ovarian microvascular endothelial cells (HOMEC). Thus, the novel preparative procedure described now allows the generation of HOMEC cultures from readily available material resulting from IVF procedures.

Cells, Cultured↗
PubMed2005

The effect of 6-mercaptopurine on early human placental explants.

BACKGROUND: 6-mercaptopurine (6-MP) is an antineoplastic and immunosuppressive drug. Recently, more women have received this drug during pregnancy. Animal studies have shown that 6-MP has deleterious effects on the fetus, while human data include prematurity, intrauterine growth restriction, low birth weight and malformations that occur especially when the drug is administered in the first trimester of pregnancy. OBJECTIVES: To study the effects of 6-MP on cellular functions of human trophoblast explants. METHODS: Human placental explants (5.5-9 weeks gestational age), that were grown on matrigel, were exposed to medium containing 6-MP for 5 days. Medium alone served as control. Extravillous trophoblast (EVT) cell migration assessment was performed by visual observation. Analysis of proliferating events of the trophoblast cells was assessed by immunohistochemical examination. Apoptosis was analyzed by Tunnel procedure and by anti-caspase 3 staining and hormone level by enzyme-linked immunosorbent assay. RESULTS: 6-MP inhibited migration of EVT cells from the villi to the matrigel with a lower proliferation rate and increased apoptosis of cytotrophoblast cells compared to controls. However, no significant effect of 6-MP on hormone levels was observed. CONCLUSIONS: 6-MP inhibited migration and proliferation of trophoblast cells in first-trimester human placental explant culture.

Antimetabolites, Antineoplastic↗