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PubMed1994

The integrin alpha v gene: identification and characterization of the promoter region.

We isolated a 15.5 kilobase pair DNA fragment that contains the 5' end of the human vitronectin receptor alpha subunit (alpha v) gene. The nucleotide sequence of the 5' flanking region, first exon and part of the first intron of the alpha v gene was determined. The sequence showed that the 5' end of the alpha v gene lies within a CpG island. The transcriptional initiation site was mapped 169 base pairs upstream of the alpha v translational initiation site. The 5' flanking region of the alpha v gene does not contain TATA or InR transcriptional control elements but does contain four Sp1 binding sites, two Ets binding sites and one GATA binding site. The identified alpha v gene 5' flanking region directed the expression of human growth hormone in transfected HeLa cells. Successive deletions of the 5' flanking region demonstrated a 222 bp region that exerts a strong positive effect on alpha v promoter activity.

Amino Acid Sequence↗

PubMed2005

Expression of integrin alpha5 and integrin beta4 and their extracellular ligands fibronectin and laminin in human decidua during early pregnancy and its sex steroid-mediated regulation.

The reorganization of the human endometrium is termed decidualization, which includes endometrial cell proliferation, differentiation, integrin switching and extracellular matrix (ECM) remodeling during early pregnancy. The present study aimed to investigate distribution patterns, staining intensity and sex steroid-mediated regulation of integrin alpha5 (CD49e), integrin beta4 (CD49f) expression and their ligands fibronectin and laminin during decidualization. Human tissue samples were evaluated in two groups, those collected in early days and those collected in advanced days of the first trimester. Correlating immunostaining was found between laminin and integrin beta4, and between fibronectin and integrin alpha5. The expression of fibronectin was higher than that of laminin in the early days (p < 0.05). Temporal and spatial immunostaining of integrin beta4 and alpha5 in the apical pole of luminal and glandular cells was observed as pregnancy progressed (p < 0.05). In vitro results showed that human chorionic gonadotropin (hCG) stimulated laminin expression, downregulated integrin beta4 expression, whereas estradiol decreased fibronectin expression by Ishikawa cells. hCG suppressed fibronectin expression in endometrial stromal cells in culture. Our results suggest that fibronectin is responsible for induction of decidual cell differentiation, and different temporal and spatial expression of the integrins may play a role in implantation. Our in vitro results suggest that regulation of extracellular matrix remodeling and integrin switching are at least partially regulated by reproductive hormones.

Adult↗

PubMed2006

Endothelial microparticles and platelet and leukocyte activation in patients with the metabolic syndrome.

Accumulating evidence has shown a strong association between the metabolic syndrome (MS) and a chronic inflammatory state predisposing to atherosclerosis. We investigated leukocyte, platelet, and endothelial activation markers and cellular interactions in 33 patients with the MS and 25 healthy controls. Using flow cytometry, we measured: (1)P-selectin expression in platelets; (2) platelet microparticles identified by CD31 expression; (3) endothelial microparticles (EMPs) identified by expression of CD31 (EMP(31)), CD62E (EMP(62E)), and CD51 (EMP(51)); (4) conjugates of leukocytes with platelet microparticles/platelets and with EMPs identified by CD54 (EMP(54)); and (5) CD11b expression in leukocytes. Patients with the MS had markedly elevated EMP(31), although EMP(62E) levels were normal, suggesting that EMP(31) levels were increased because of endothelial cell apoptosis, rather than activation. EMP(51), EMP(54)-lymphocyte conjugates, platelet expression of P-selectin, CD11b expression in leukocytes, and platelet-lymphocyte conjugates were also increased in patients with the MS. Platelet-leukocyte conjugates correlated with leukocyte activation, suggesting that platelet binding to leukocytes regulates leukocyte activation in vivo. In conclusion, our data demonstrate endothelial cell microparticle release, platelet and leukocyte activation, and increased binding of EMPs and platelets to leukocytes in patients with the MS.

Biomarkers↗

PubMed2004

Expression, production, and characterization of full-length vitronectin in Escherichia coli.

Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GST/VN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GST/VN was recovered from 1L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies.

Animals↗

PubMed2005

Expression, purification, and characterization of a neovasculature targeted rmhTNF-alpha in Escherichia coli.

The tumor vasculature is a suitable target for cancer treatment. RGD-4C (CDCRGDCFC) peptide can bind to human alphav integrins, which are known to be selectively expressed in human tumor blood vessels. Some studies showed that coupling anticancer drugs or peptides to the RGD peptides yielded compounds with increased efficacy against tumors and lowered toxicity to normal tissues in mice. TNF-alpha mutant (rmhTNF-alpha) that we previously constructed has been proved to have stronger antitumor effect compared with TNF-alpha. To increase antitumor effect and lower toxicity of rmhTNF-alpha, we coupled RGD4C to the N-terminal of rmhTNF-alpha (termed RGD4C-rmhTNF) and expressed RGD4C-rmhTNF in Escherichia coli. Here, we describe the expression, purification, and characterization of RGD4C-rmhTNF.

Animals↗

PubMed2004

Extracellular fragment of brain-specific angiogenesis inhibitor 1 suppresses endothelial cell proliferation by blocking alphavbeta5 integrin.

Brain-specific angiogenesis inhibitor 1 (BAI1) is a transmembrane protein with anti-angiogenic activity. The mechanisms underlying BAI1 activity are unknown. In this study, we found that overexpression of BAI1 increased cell death in human umbilical vein endothelial cells (HUVECs) and, to a lesser degree, in SHSY5Y and U343 cells. Conditioned medium from BAI1-transfected U343 cells inhibited proliferation of HUVECs, and this effect was neutralized by addition of anti-BAI1 serum. The conditioned medium contained four cleavage products of the BAI1 extracellular domain. BAI1's middle extracellular region containing five thrombospondin type 1 repeats (BAI1-TSR) was sufficient for BAI1's antiproliferative effect on HUVECs. BAI1's action on HUVECs was blocked by anti-alpha(v) integrin, but not by anti-CD36 antibody treatment. Introduction of alpha(v)beta(5) integrin into HEK293 cells rendered them susceptible to cell death by BAI1, and BAI1-TSR bound with alpha(v)beta(5) integrin, but not to alpha(v)beta(3) integrin in brain tissue. Fluorescent BAI1-TSR colocalized with alpha(v)beta(5) integrin in HUVECs. Together, our results indicate that BAI1 has antiproliferative action on surrounding endothelial cells by blocking alpha(v)beta(5) integrin, and its active region is BAI1-TSR. BAI1-TSR could be valuable for regulating brain angiogenesis.

Angiogenesis Inhibitors↗

PubMed2006

Alpha-V-dependent outside-in signaling is required for the regulation of CD44 surface expression, MMP-2 secretion, and cell migration by osteopontin in human melanoma cells.

The level of integrin alpha(v)beta3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp(60c-Src) kinase activity associated with integrin alpha(v)beta3 in melanoma cells (M21) treated with soluble OPN. pp(60c-Src) kinase activity was not observed in melanoma cells expressing alpha(v) that lacks the cytoplasmic domain (alpha(v)995). Results of the current study demonstrate that the amino acid sequence '995RPPQEEQERE1004' in the beta-turn of alpha(v) chain is required for the interaction of pp(60c-Src). Our results suggest that the beta-turn of alpha(v) chain may be indispensable for alpha(v)-associated signaling complex formation and outside-in signaling. To further analyze the alpha(v)beta3 signaling in melanoma cells, we over expressed OPN in M21 cells (M21/OPN). CD44 surface expression and MMP-2 activity in the conditioned medium were increased to a greater extent in M21/OPN cells as compared with M21 or alpha(v)995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to alpha(v) and MMP-2. Our findings suggest that the increase in MMP-2 activity is integrin-dependent as MMP-2 activity is reduced in cells treated with an inhibitor to alpha(v) or in alpha(v)995 cells expressing mutant alpha(v).

Actins↗

PubMed1998

Endothelial cell integrin laminin receptor expression in multiple sclerosis lesions.

Laminin, a major glycoprotein component of vessel basement membranes, is recognized by beta1- and beta3-integrins expressed on endothelial cells. To determine how endothelial cell integrins might function in multiple sclerosis (MS) lesions, integrin laminin receptors and laminin were analyzed in central nervous system samples from MS patients and controls by immunohistochemistry. In active MS lesions, endothelial cell VLA-6 and beta1 subunits were decreased compared to controls whereas alpha(v) subunit and VLA-1 were increased. In chronic inactive lesions beta1, VLA-6 and alpha(v) were the same as controls but VLA-1 remained increased. Alpha3 subunit was constant in all samples. By immunoelectron microscopy VLA-1, VLA-6, beta1, and laminin were distributed throughout endothelial cells; alpha(v) was adjacent to and on luminal surfaces; alpha(v) and VLA-1 were on intercellular junctions. These results indicate distinct regulation and functions of these integrins in different lesion stages. In active lesions decreased endothelial cell beta1/VLA-6 could result in their detachment from laminin thereby facilitating leukocyte transvascular migration and blood-brain barrier breakdown. Alpha(v) and VLA-1 on intercellular junctions may participate in re-establishing vessel integrity after leukocyte migration. Luminal surface alpha(v) also likely binds intraluminal ligands and cells. In chronic inactive plaques persistently elevated endothelial cell VLA-1 correlates with long-standing endothelial cell and blood-brain barrier dysfunction.

Adolescent↗

PubMed2001

Patterns of integrin expression in a human mandibular explant model of osteoblast differentiation.

Cell-matrix interaction is crucial in regulating osteoblast differentiation and function. These interactions are themselves regulated, at least in part, by integrins. Although there are some data from mammalian models, few studies have compared integrin expression at different stages of the osteoblast lineage. Here, primary human mandibular osteoblast cultures were grown in the presence of epidermal growth factor (EGF), giving a proliferative, less differentiated phenotype, or of vitamin D(3) and hydrocortisone (D+Hc), giving a more differentiated phenotype. These cultures were compared with those of cells prepared in the absence of EGF or D+Hc by fluorescence-activated cell sorter using a panel of monoclonal antibodies to specific integrin heterodimers. To provide in vivo correlation, the same panel of antibodies was used to stain fresh-frozen, undemineralised sections of human mandibular bone. Under baseline conditions the alpha(3), alpha(5), alpha(v), alpha(v)beta(3), beta(3) and beta(1) integrin subunits were expressed strongly by the cells, with low-level expression of the alpha(1), alpha(2) and alpha(4) subunits. In the presence of EGF there was increased alpha(2) expression. With D+Hc, alpha(3) and alpha(5) expression was elevated. Immunohistochemical analysis demonstrated alpha(2), alpha(3), alpha(5), alpha(v)beta(3), beta(1) and beta(3) subunits in cells of the osteoblast lineage; alpha(2) staining was restricted to cells close to the bone surface whilst alpha(v)beta(3) and beta(3) were most frequently localised in the osteocytes. The results provide evidence that cells at successive stages of the osteoblast lineage show different patterns of integrin expression. These integrins may be important in cell-matrix interactions leading to osteoblast differentiation.

Antibodies, Monoclonal↗

PubMed2003

Specific alpha v integrin receptors modulate K1735 murine melanoma cell behavior.

Expression of beta 3 integrins is increased in invasive melanoma. In this study we show that K1735 cell proliferation is enhanced by the expression of either beta 3 or a constitutively active Src. We investigated possible modulators of FN matrix assembly and found that matrix metalloproteinase 2 (MMP2) was activated by alpha v beta 3. alpha v beta 3 integrin was localized to focal contacts whereas alpha v beta 5 was peripherally distributed. MMP2 was also activated by expression of CASrc. MMP2 activation inversely correlated with FN matrix assembly, in that it dramatically reduced the organization of a FN matrix. K1735 cell migration on VN and invasion through a reconstituted basement membrane were decreased in the presence of anti-MMP2 antibodies. These results demonstrate that the expression of the alpha v beta 3 complex modulates melanoma cell behavior including activation of Src, organization of the cytoskeleton, assembly of the extracellular matrix, cell motility, and activation of MMP2.

Animals↗

PubMed2001

Differential expression of integrin alpha v and beta 3 in serosal tissue of human intraperitoneal organs and adhesion.

OBJECTIVE: To assess the expression of integrin alpha v and beta 3 in the serosal tissue of intraperitoneal organs and adhesions in persons with and without adhesions. DESIGN: Prospective study. SETTING: Academic research centers. PATIENT(S): Fifty-seven patients undergoing abdominal or pelvic surgery. MAIN OUTCOME MEASURE(S): Integrin alpha v and beta 3 messenger RNA (mRNA) expression was evaluated by quantitative reverse transcription polymerase chain reaction. RESULT(S): The serosal tissue of the parietal peritoneum, uterus, fallopian tubes, ovary, and the large and small bowel, as well as peritoneal adhesions, skin, fascia, subcutaneous tissue, and omentum, expresses integrin alpha v and beta 3 mRNA. The level of alpha v and beta 3 mRNA expression varied among these tissues; expression of the former substance was highest in uterine serosa and lowest in the skin and small bowel, and expression of the latter substance was highest in the fallopian tubes and skin and lowest in the uterine serosa. Parietal peritoneum and adhesions express equal levels of integrin alpha v; however, integrin beta 3 expression was >100-fold lower in adhesions than in peritoneum. The level of integrin beta 3 expression in omentum, small and large bowels, and subcutaneous tissue was 100-fold to 10,000-fold lower than in other tissues. CONCLUSION(S): Serosal tissue of peritoneal organs and adhesions express variable levels of integrin alpha v and beta 3 mRNA. On the basis of such variation and the knowledge that tissue injury alters local integrin expression, integrins may play a key role in adhesion development, particularly in tissue with higher integrin expression.

Adult↗

PubMed2002

Transforming growth factor-beta1 modulates expression of adhesion and cytoskeletal proteins in human peritoneal fibroblasts.

OBJECTIVE: To determine the effects of TGF-beta1 on the expression of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and on vinculin, and F-actin in human peritoneal fibroblasts. DESIGN: Descriptive study using cell culture, reverse transcriptase-polymerase chain reaction (RT-PCR), and immunofluorescent and confocal microscopy. SETTING: Academic medical center. PATIENT(S): Gynecological surgery patients. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Effects of TGF-beta1 on the steady state levels of alpha5, alpha(v), and alpha6 integrin transcripts were examined in the normal peritoneal fibroblasts using RT-PCR. Expression levels of the alpha1, alpha2, alpha5, alpha(v), and alpha6 integrin subunits and of F-actin were measured by immunofluorescent microscopy. The distribution pattern of the integrin subunits, vinculin, and F-actin were examined using confocal microscopy. RESULT(S): TGF-beta1 significantly up-regulated the expression levels of the alpha5, alpha(v), and alpha6 integrin subunits and modulated their expression pattern, resulting in relatively higher levels of these subunits in the focal contacts of peritoneal fibroblasts. It allocated vinculin expression primarily to the focal contacts of cells and caused distortion of F-actin structure. The transcript levels of the alpha5, alpha(v), and alpha6 integrin subunits were not altered by TGF-beta1 treatment. CONCLUSION(S): TGF-beta1 may promote postoperative adhesion formation by inducing the migration of peritoneal fibroblasts by altering the expression levels and patterns of specific integrin subunits, vinculin, and F-actin.

Actins↗

PubMed1997

Efficient adenoviral-mediated murine neonatal small intestinal gene transfer is dependent on alpha(v) integrin expression.

BACKGROUND/PURPOSE: Clinical application of gene therapy for patients who have inflammatory bowel disease or short bowel syndrome will require the development of new strategies to improve the efficiency of small intestinal gene transfer. Previously, the authors developed a method for adenoviral-mediated small intestinal gene transfer in vivo in neonatal and adult mice. The present study evaluates the hypothesis that the integrins alpha(v)beta3 and alpha(v)beta5, the secondary receptors for adenoviral internalization, play a facilitative role in neonatal murine adenoviral-mediated small intestinal gene transfer. METHODS: Immunohistochemical techniques identified the integrin alpha(v)beta3 and the integrin subcomponents alpha(v), beta3, and beta5 in neonatal and adult small intestine. The effects of integrin receptor antagonists on transgene expression was also studied in our neonatal model of adenoviral-mediated small intestinal gene transfer in vivo. RESULTS: Gene transfer was significantly decreased by the addition of integrin receptor antagonists versus control peptide. Integrin alpha(v)beta3 and integrin subcomponent alpha(v), beta3, and beta5 are expressed in neonatal and adult small intestine. Integrin antagonists administered simultaneously blocked efficient adenoviral-mediated neonatal small intestinal gene transfer in vivo compared with control peptide. CONCLUSION: Strategies to upregulate integrin expression may improve adenoviral-mediated small intestinal gene transfer.

Adenoviruses, Human↗

PubMed2001

Phagocytosis in vitro of polyethylene glycol-modified liposome-encapsulated hemoglobin by human peripheral blood monocytes plus macrophages through scavenger receptors.

Liposome-encapsulated hemoglobin (LEH), a candidate for red blood cell substitute, is cleared from circulation primarily by the phagocytic system, most likely after opsonization of the vesicles by immunoproteins, particularly complement components. Although modification of LEH by polyethylene glycol (PEG) derivatives prolongs its half-life by blocking the opsonization, the half-life is still short as compared with that of red blood cell components. Therefore, this study was performed to elucidate the opsonin-independent mechanisms that regulate phagocytosis of Neo Red Cell (NRC), a PEG-modified LEH, in culture. PKH67 was used as a fluorescence marker, allowing the quantitation of the phagocytosis of NRC by peripheral blood monocytes plus macrophages. The phagocytosis of PKH67-labeled NRC was inhibited by the addition of an excess of unlabeled NRC, indicating that the phagocytosis of PKH67-labeled NRC is specific to NRC, but not to PKH67. The phagocytosis of NRC was blocked about 70% by anti-CD14, 60% by anti-CD36 and 30% by anti-CD51/61 (vitronectin receptor, alpha(v)beta3). These results provided evidence of an opsonin-independent pathway for the phagocytosis of PEG-modified LEH.

Antigens, CD↗

PubMed1997

Immunolocalization of integrins in the normal lung and in pulmonary carcinomas.

Cryosections of normal adult lung (n = 7) and pulmonary epithelial tumors, including squamous (n = 8), adeno (n = 8), bronchioloalveolar (n = 5), and large cell (n = 4) carcinomas (SCC, ACC, BAC, LCC), carcinoids (Cd, n = 7), and neuroendocrine carcinomas (NEC) of variable grades (n = 14) were immunostained by the avidin-biotin peroxidase (ABC) method with monoclonal antibodies to the alpha1-6 and alpha(v) and the beta1-4 integrin subunits. Normal adult alveolar septae showed variably intense immunoreactivity for alpha1,3,6 and beta1, whereas reactions for alpha5 and alpha(v) were weaker and uneven; the remaining integrin subunits were not detected. Bronchial and bronchiolar epithelium showed variably intense staining for alpha2.3,6,v and beta1,4. Reactions were often, though not invariably, basally polarized. SCC, ADC, and LCC showed variably intense reactions for alpha2.3,6,v and beta1,4. BAC were strongly and uniformly stained for alpha1.3 and beta1. In Cd, alpha1,2,3,v and beta1 reactions were noted, whereas in NEC, weak alpha1,3 and beta1 staining was detected with only traces of alpha6 and alpha(v). We conclude that alveolar epithelial cells do not express the hemidesmosome-associated, laminin-binding integrin alpha6beta4 of the bronchial epithelium but rather the alpha1beta1 and alpha3beta1, collagen IV, and laminin receptors, respectively. SCC, ADC, and sampled LCC express an integrin repertory qualitatively similar to that of the bronchial epithelium. Distinct from the latter, the integrin repertory of BAC parallels that of the alveolar epithelium by its strong expression of the multipotential alpha1beta1 and alpha3beta1 integrins. NEC tumors do not display the laminin receptors alpha6beta4 and alpha6beta1 shown by SCC and ADC but express instead alpha1beta1, a collagen IV-laminin receptor rarely found in epithelial neoplasms except for BAC. In NEC tumors, integrins, especially alpha2, decrease with dedifferentiation. Notably distinct from epithelial mesotheliomas, the major fibronectin-binding integrin alpha5beta1 was not found in any type of lung carcinoma.

Adenocarcinoma↗

PubMed1996

The adaptor protein Shc couples a class of integrins to the control of cell cycle progression.

We provide evidence that a class of integrins combines with the adaptor Shc and thereby with Grb2. Coimmunoprecipitation and mutagenesis experiments indicate that the recruitment of Shc is specified by the extracellular or transmembrane domain of integrin alpha subunit and suggest that this process is mediated by caveolin. Mutagenesis and dominant-negative inhibition studies reveal that Shc is necessary and sufficient for activation of the MAP kinase pathway in response to integrin ligation. Mitogens and Shc-activating integrins cooperate to promote transcription from the Fos serum response element and transit through G1. In contrast, adhesion mediated by integrins not linked to Shc results in cell cycle arrest and apoptosis even in presence of mitogens. These findings indicate that the association of specific integrins with Shc regulates cell survival and cell cycle progression.

3T3 Cells↗

PubMed1997

Cross-reactivity of monoclonal antibodies to defined human leucocyte differentiation antigens with bovine cells.

Thirty-seven subpanels of monoclonal antibodies (mAbs) included within the Vth International Workshop on Human Leucocyte Differentiation Antigens (Vth Workshop) were assayed for reactivity with bovine peripheral blood leucocytes. Sixty-five of the 772 mAbs (8.4%) stained bovine cells. mAbs from each of the 27 different CD groups that contained a mAb reacting with cattle were further investigated to compare the cellular expression of the antigen in cattle with that reported for the different CD antigens in humans. Two-colour immunofluorescence staining of the Vth Workshop mAbs against characterized bovine leucocyte subpopulation markers that identified monocytes, B cells, CD4, CD8 and WC1 +T cells were used for these analyses. Eighteen of the mAbs to different human CD antigens (CD11a, CD14, CD18, CD21, CD27, CD29, CD49a, CD49b, CD49d, CD49e, CD51, CD61, CD62L, CD62P, CD63, CDw78, CD98, CD100) stained bovine antigens with an almost identical cellular distribution to that reported in humans. This implies that these mAb react with the homologous cattle molecules. Nine mAbs (CD35, CD37, CD49c, CD50, CD54, CD66, CD81, CD88, CD102) stained bovine cells but the cellular distribution of the bovine antigen was different to that reported in humans implying either a different cellular distribution for these antigens in cattle or a reaction with a different molecule. The investigation has allowed the identification of several bovine homologues of human CD antigens that have not been previously defined in cattle and the cross-reacting mAbs will be valuable reagents for future investigations of bovine immunology.

Animals↗

PubMed2002

Primary human cells differ in their susceptibility to rAAV-2-mediated gene transfer and duration of reporter gene expression.

The susceptibility of a variety of different primary tissues was examined to long-term transduction with recombinant adeno-associated virus type 2 (rAAV-2) and factors influencing the transduction efficiency. In contrast to others using cell lines and animal models, emphasis was placed on the use of primary human cells. Enhanced green fluorescent protein (EGFP) marker gene expression was examined using fluorescence-activated cell sorting analysis. The most effective target cells for rAAV-2-mediated gene transfer were bronchial epithelial, artery endothelial as well as smooth and skeletal muscle cells with mean transduction rates ranging from 34.3 to 81.6%. Lower transduction rates between 4.3 and 19.5% were found in chondrocytes, dermal papilla follicle epithelial cells and fibroblasts. No transduction was observed in melanocytes, granulocyte colony-stimulating factor (G-CSF)-mobilized CD34(+) cells or malignant CD19(+) cells from patients with chronic lymphocytic leukemia. A proportion of EGFP-expressing skeletal muscle and smooth muscle cells was maintained over a period of 6 weeks after transduction (42.7+/-5.4 and 67.1+/-0.9%, respectively). Interestingly, among hair follicle epithelial cells the proportion of transduced cells increased from 8+/-0.5 to 36+/-7.7% in the course of 6 weeks. In contrast, for endothelial cells, bronchial epithelial cells and fibroblasts, a rapid decline in the number of EGFP expressing cells were noted. An inverse relationship between the proportion of cells in G2/M phase of cell cycle and long-term gene expression was observed. All rAAV-2 susceptible primary cells expressed FGFR-1 and the alphaV integrin consistent with their role as co-receptors for AAV-2. In conclusion, AAV-2 is a suitable vector system for transduction and evaluation of functional effects of long-term gene expression in primary human muscle and hair follicle cells.

Cell Line↗