Coexpression of the Genes for Platelet-Derived Growth Factor and Its Receptor in Human T-Cell Lines Infected with HTLV-I

Abstract

Several HTLV-I-infected T-cell lines express the two genes encoding platelet-derived growth factor (PDGF). Therefore, we examined the question of a possible self-stimulatory mechanism of proliferation involving PDGF in these cells. Using a nucleic acid probe and an antibody specific for the PDGF-B receptor (PDGFR-B), we examined four established human T-cell lines infected with HTLV-I, as well as several noninfected T-cell lines for expression of the PDGF-B receptor. Previous reports indicate that lymphocytes do not display receptors for PDGF; our results show that two noninfected T-cell lines (HUT-78, CCRF-HSB-2) expressed the canonical 5.5-kb PDGFR mRNA, and two HTLV-I-infected T-cell lines (C10/ MJ, MT-2) expressed a novel PDGFR mRNA of 4.8 kb. Concomitantly, the cell lines expressing PDGFR mRNA also synthesize PDGFR proteins immunoprecipitated by the antibody to PDGFR-B. Differences were observed in the molecular weight of PDGFR molecules immunoprecipitated from uninfected and HTLV-infected T-cells. Immunofluorescence studies demonstrated that the PDGFR-B proteins are localized primarily on the cell external membrane. The results suggest that the HTLV-I-infected T-cells acquire an autostimulatory mechanism of cell proliferation that involves PDGF.     

Expression of Keratinocyte Growth Factor and Its Receptor in Rat Tracheal Cartilage: Possible Involvement in Wound Healing of the Damaged Cartilage

Keratinocyte growth factor (KGF) is involved in the development and regeneration of a variety of tissues. To clarify the role of KGF in cartilage wound healing, we examined the expression of KGF and its receptor (KGFR) immunohistochemically in the wound healing area of rat tracheal cartilage, and the direct effect of recombinant KGF on the proliferation and differentiation of primary cultures of rat chondrocytes. KGF was found in the cytoplasm of both chondrocytes and perichondrial cells. On the other hand, KGFR was detected only in the plasma membrane of chondrocytes. Although the expression of KGF was similar in the cartilage and perichondrial area before and after injury, KGFR expression was induced after injury and limited to proliferating chondrocytes. The staining pattern of KGF and KGFR was same in the mature and the immature rat tracheal cartilage. Moreover, in vitro experiments using primary cultured chondrocytes revealed that KGF at 200 ng/ml significantly increased the number of chondrocytes (~1.5-fold), and significantly reduced acid mucopolysaccharide production. These results indicate that KGF stimulates chondrocyte proliferation, suggesting that KGF could therapeutically modulate the wound healing process in the tracheal cartilage.     

髌韧带血供及其在重建膝交叉韧带中的意义

目的：探讨髌韧带血供的特点,为临床交叉韧带移植重建提供应用解剖学资料。方法：通过对成人和胎儿下肢标本经股动脉红色乳胶灌注并解剖观察,以及胎儿墨汁灌注组织透明和组织切片等方法,观察髌韧带的动脉来源、分布特点,并测量胎儿韧带内微血管密度。结果：髌韧带的动脉来自膝下外动脉、胫前返动脉、膝降动脉和膝下内侧动脉的分支;胎儿髌韧带不同区域微血管密度不同,以韧带中心部位密度最低。结论：髌韧带中心部为相对乏血管区,对以髌韧带为替代物行交叉韧带重建有重要的临床意义。     

Time Course of Cell Proliferation in Rat Liver in the Early Postnatal Ontogeny and Role of Epidermal Growth Factor in Organization of Proliferative Regimen

sCircadian rhythm of DNA synthesis, mitotic activity, and duration of mitosis in rat liver were studied on days 3, 7, and 12 of life. Age-associated differences in the rhythmic parameters of these characteristics were detected. Epidermal growth factor plays an important role in the formation of cell proliferation rhythm in the early postnatal ontogeny and in the formation of proliferative hepatocyte pool.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 5, pp. 585–588, May, 2005     

Differential Endothelial Migration and Proliferation to Basic Fibroblast Growth Factor and Vascular Endothelial Growth Factor

Abstract

Neovascularization is a feature of a variety of pathological processes. We compared the characteristics of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) on migration and proliferation of human umbilical vein endothelium (HUVEC). Both VEGF and bFGF induced endothelial cell migration at similar concentrations (½ max. VEGF = ～1.0 ng/ml, bFGF = ～5.0 ng/ml). However, VEGF-stimulated migration was two-fold greater than bFGF at 1 and 10 ng/ml (p < 0.05). In contrast, bFGF induced proliferation four-fold more effectively than VEGF (½ max. 1 ng/ml and 1.4 ng/ ml respectively). Checkerboard migration assays for bFGF showed a predominantly chemokinetic pattern, whereas VEGF was predominantly chemotactic. VEGF and bFGF were not synergistic in monolayer proliferation and migration assays. Three angiogenesis inhibitors, alpha-interferon, TNP-470, and platelet factor-4, inhibited VEGF and bFGF induced cell migration. These results indicate that VEGF and bFGF are chemoattractants that stimulate endothelial migration by different mechanisms and that both can be inhibited by known angiogenesis inhibitors.     

Angiotensin Receptor Blockers and Statins Could Alleviate Atrial Fibrosis via Regulating Platelet-Derived Growth Factor/Rac1 /Nuclear Factor-Kappa B Axis

Aims: To investigate whether the administration of renin-angiotensin system (RAS) inhibitors and statins could alleviate atrial fibrosis via platelet-derived growth factor (PDGF)/Rac1 /nuclear factor-kappa B (NF-κB) axis.Methods and Results: In human left atrium, the degree of atrial fibrosis, as well as the expression levels of PDGF, Rac1 and NF-κB increased 1.5 to 2.9 folds in patients with atrial fibrillation compared to that with sinus rhythm, (P<0.0001). There were strongly positive correlations between angiotensin II (Ang II) or procollagen type III-alpha-1 (COL3A1) with PDGF, Rac1, NF-κB, and among PDGF, Rac1 and NF-κB (all P<0.05). At 3 weeks after the transverse aorta constriction (TAC) operation in rat model and with intervention of irbesartan or/and simvastatin, the collagen volume fraction (CVF) and atrial natriuretic peptide (ANP) values respectively increased 6-folds and 3.5-folds in the TAC group compared to SHAM group (P<0.0001), but these levels decreased by 16% to 63% with following drug intervention (all P<0.0001), the combined treatment was the lowest. Accordingly, the expression levels of PDGF (3-folds), Rac1 (1.6-folds), NF-κB (7-folds) and AngII (12-folds) significantly increased in the TAC group compared to the SHAM group, and these levels were also reduced by 25% to 64% with following drug intervention. The highest reduction could be seen after treatment with irbesartan and simvastatin in combination (all P<0.001).There were strongly positive correlations between AngII or CVF with PDGF, Rac1, NF-κB, and among PDGF, Rac1 and NF-κB (all P<0.05).Conclusions: Irbesartan or/and simvastatin can improve atrial fibrosis by regulating PDGF/Rac1/NF-κB axis.     

Tendon matrix composition and turnover in relation to functional requirements

Tendons are dense regular connective tissue structures that are defined based on their anatomical position of connecting muscle to bone. Despite these obvious commons features tendons from different locations within the body show remarkable variation in terms of their morphological, molecular and mechanical properties which relates to their specialized function. An appreciation of these differences is necessary to understand all aspects of tendon biology in health and disease. In our work, we have used a combination of mechanical assessment, histological measurements and molecular analysis of matrix in functionally distinct tendons to determine relationships between function and structure. We have found significant differences in material and molecular properties between spring-like tendons that are subjected to high strains during locomotion and positional tendons which are subjected to much lower strains. Furthermore, we have data to suggest that not only is the matrix composition different but also the ability of cells to synthesize and degrade the matrix (matrix turnover) varies between tendon types. We propose that these differences relate to the magnitude of strain that the tendon experiences during normal activities in life. Tendon cells may be preprogrammed during embryological development for the strain they will encounter in life or may simply respond to the particular strain environment they are subjected to. The elucidation of controlling mechanisms resulting in tendon cell specialization will have important consequences for cell based therapies and engineering strategies to repair damaged tendons.     

Expression of p75NGFR,a Proliferative and Basal Cell Marker,in the Buccal Mucosa Epithelium during Re-epithelialization

We investigated the expression of p75^NGFR, a proliferative and basal cell marker, in the mouse buccal mucosa epithelium during wound healing in order to elucidate the role of epithelial stem cells. Epithelial defects were generated in the epithelium of the buccal mucosa of 6-week-old mice using CO₂ laser irradiation. BrdU was immediately administered to mice following laser irradiation. They were then sacrificed after 1, 3, 7, and 14 days. Paraffin sections were prepared and the irradiated areas were analyzed using immunohistochemistry with anti-p75^NGFR, BrdU, PCNA, and CK14 antibodies. During re-epithelialization, PCNA (–)/p75^NGFR (+) cells extended to the wound, which then closed, whereas PCNA (+)/p75^NGFR (+) cells were not observed at the edge of the wound. In addition, p75^NGFR (–)/CK14 (+), which reflected the presence of post-mitotic differentiating cells, was observed in the supra-basal layers of the extended epithelium. BrdU (+)/p75^NGFR (+), which reflected the presence of epithelial stem cells, was detected sparsely in buccal basal epithelial cells after healing, and disappeared after 7 days. These results suggest that p75^NGFR (+) keratinocytes are localized in the basal layer, which contains oral epithelial stem cells, and retain the ability to proliferate in order to regenerate the buccal mucosal epithelium.     

Gene Expression of Tendon Collagens and Tenocyte Markers in Long-Term Monolayer and High-Density Cultures of Rat Tenocytes

As a result of repeated movement, tendons are functionally open to traumas. According to this situation, tenocytes have already been used for tissue engineering therapies. It has been reported that long-term monolayer (ML) culture of tenocytes may lead to a phenotypic drift within passages. Depending on our previously published work, it is clearly demonstrated that high-density (HD) culture improves cell growth and differentiation of tenocytes. However, it is not yet established if HD favors the differentiated state during long-term culture. Therefore, we compared the differences in gene expression of tendon collagens and tendon markers of tenocytes from long-term ML and HD culture conditions by quantitative, real-time polymerase chain reaction (QRT-PCR) for over a period of 3 weeks. COLI, COLIII, COLV, Scx, and Tnmd were target genes as the major matrix constituents of tendons as well as being involved in matrix integrity and tenocyte phenotype. According to our results, tenocytes in HD culture synthesized less amounts of COLIII, COLV, and Tnmd, and dependent on the investigation time point, higher amounts of Scx. We consider that tenocytes produced in HD culture system may not provide sufficient efficiency during tissue engineering approaches. By the fact that most molecules showed significantly higher expression profiles in ML culture condition, it is suggested that culture and passage in ML should be taken into consideration for further tissue engineering approaches to maintain a phenotype with less amount of drift.     

Sex-Steroid Regulation of Relaxin Receptor Isoforms (RXFP1 & RXFP2) Expression in the Patellar Tendon and Lateral Collateral Ligament of Female WKY Rats

The incidence of non-contact knee injury was found higher in female than in male and is related to the phases of the menstrual cycle. This raised the possibility that female sex-steroids are involved in the mechanism underlying this injury via affecting the expression of the receptors for relaxin, a peptide hormone known to modulate ligament laxity. Therefore, this study aims to investigate the effect of sex-steroids on relaxin receptor isoforms (RXFP1 & RXFP2) expression in the ligaments and tendons of the knee. Methods: Ovariectomized adult female WKY rats were treated with different doses of estrogen (0.2, 2, 20 μg/kg), progesterone (4mg) and testosterone (125 & 250μg/kg) for three consecutive days. At the end of the treatment, the animals were sacrificed and the patellar tendon and lateral collateral ligament were harvested for mRNA and protein expression analyses by Real Time PCR and Western blotting respectively. Results: RXFP1, the main isoform expressed in these knee structures and RXFP2 showed a dose-dependent increase in expression with estrogen. Progesterone treatment resulted in an increase while testosterone caused a dose-dependent decrease in the mRNA and protein expression of both relaxin receptor isoforms. Discussion: Progesterone and high dose estrogen up-regulate while testosterone down-regulates RXFP1 and RXFP2 expression in the patellar tendon and lateral collateral ligament of rat''s knee. Conclusion: Relaxin receptor isoforms up-regulation by progesterone and high dose estrogen could provide the basis for the reported increase in knee laxity while down-regulation of these receptor isoforms by testosterone could explain low incidence of non-contact knee injury in male.     

结缔组织生长因子siRNA对大鼠血管平滑肌细胞增殖和迁移的影响

目的观察结缔组织生长因子(CTGF)siRNA对CTGF表达及对大鼠血管平滑肌细胞(VSMC)增殖和迁移的影响。方法培养大鼠血管平滑肌细胞,用脂质体法将抗CTGF483siRNA进行转染,Western blot法检测转染效果,MTT法及划痕实验测定对VSMC增殖和迁移的影响。结果转染CTGF483siRNA组的CTGF表达明显低于未转染组,MTT法显示转染CTGF483siRNA后VSMCs增殖减慢,划痕试验证实转染CTGF483siRNA后VSMCs迁移受到抑制。结论CTGFsiRNA可以抑制VSMC增殖和迁移,有望为颈动脉粥样硬化性缺血性脑血管病的预防和治疗新靶点。     

Relationship of Keratinocyte Growth Factor and Hepatocyte Growth Factor Levels in Rat Lung Lavage Fluid to Epithelial Cell Regeneration after Bleomycin

Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are known mitogens for normal alveolar Type 2 cells in vitro and in vivo. We wished to determine whether these two growth factors are involved in lung repair after epithelial cell necrosis by determining the levels of each factor in lung lavage fluid collected serially after bleomycin-induced injury, and how these values relate specifically to proliferation of bronchiolar and alveolar epithelial cells. Rats received an intratracheal injection of 1 unit bleomycin in 0.5 ml water and were killed at intervals up to 4 weeks with 1 muCi/g tritiated thymidine injected 1 hour before death. Early necrosis of bronchiolar epithelial (BR) cells and Type 1 alveolar epithelium was followed by an increase in inflammatory cell numbers and high protein levels in bronchoalveolar lavage (BAL) fluids. In addition, the levels of KGF and HGF, measured by enzyme-linked immunosorbent assay in BAL, increased as early as 3 days and peaked at 7-14 days, when KGF was measured at 160 pg/ml (n = 50) and HGF reached 460 pg/ml (n = 40). Both values dropped sharply after 2 weeks. Epithelial cell proliferation was quantitated as percentage of labeled cells in autoradiographs of methacrylate sections. Labeling of BR cells predominated in the first week and peaked at 7% at 3 days. Type 2 cell proliferation was delayed somewhat but occurred in 3 to 10 days with a peak of 7% labeled cells at 1 week. The results demonstrate that both HGF and KGF are present in the lung in greatly increased amounts soon after bleomycin-induced epithelial cell necrosis. These high levels are associated with both BR and alveolar epithelial cell proliferation.     

PDGF、FN诱导FHL1细胞增殖的PI3K信号转导通路研究

目的探讨血小板源性生长因子（PDGF）和纤维连接蛋白（FN）诱导人胚胎肺成纤维细胞（HFL1）增殖的磷酸酰基醇-3-激酶（PI3K）信号转导通路。方法采用不同浓度的PDGF和FN分别刺激HFL1并观察PI3K抑制剂wortmannin（WMN）对PDGF、FN诱导HFL1增殖作用的影响。用四甲基偶氮唑蓝（MTT）法检测各组细胞增殖情况。结果PDGF和FN对HFL1的诱导增殖作用明显,并呈剂量依赖性升高趋势,二者均在50ng／mL时作用显著：PI3K抑制剂wortmannin可抑制PDGF和FN诱导的HFL1细胞增殖。结论PDGF和FN诱导HFL1的细胞增殖可以通过PI3K信号转导途径实现。     

Expression,Content, and Localization of Insulin-Like Growth Factor I in Human Achilles Tendon

In animals insulin-like growth factor I (IGF-I) stimulates collagen production by fibroblasts and is expressed in tendons together with its binding protein 4 (IGFBP-4). However, the presence of IGF-I and IGFBP-4 in human tendon tissue is not described. Tissue IGF-I content was examined by immunoflourometric assay, real-time PCR, and immunohistochemistry used to localize and determine expression of IGF-I and IGFBP-4 in 6 postmortem human Achilles tendons. Tendon tissue concentrations of IGF-I were found to be 0.53 ± 0.10 ng/g. Furthermore, we demonstrated that IGF-I and IGFBP-4 are localized around the tendon fibroblasts and that mRNA for IGF-I and IGFBP-4 can be determined in human tendon tissue. The present study adds support for the roles of IGF-I and IGFBP-4 in the regulation of tendon adaptive responses to mechanical loading.     

Expression of Platelet-Derived Endothelial Cell Growth Factor and its Potential Role in Up-Regulation of Angiogenesis in Scarred Kidneys Secondary to Urinary Tract Diseases

The mechanism of neovascularization secondary to renal interstitial fibrosis is not well understood. Platelet-derived endothelial cell growth factor (PD-ECGF) is known to promote angiogenesis. We examined the expression of PD-ECGF immunohistochemically in 9 normal kidneys and 26 scarred kidneys secondary to urinary tract diseases. To estimate up-regulation of angiogenesis, microvessels were counted by immunostaining endothelial cells for CD34. Immunostaining of PD-ECGF was observed in most Bowman's capsules, occasional tubules, and some interstitial mononuclear cells in normal kidneys. A remarkable increase of immunostained PD-ECGF was found in the tubules and interstitial mononuclear infiltrates in the scarred kidneys. The predominant cell type in the infiltrate was T cells (CD3(+)). The microvessel count and mean numbers of PD-ECGF(+) tubular and interstitial mononuclear cells increased with increasing interstitial fibrosis. A significant correlation was noted between microvessel count and the number of PD-ECGF(+) tubular cells (P = 0.0002) or PD-ECGF(+) interstitial mononuclear cells (P < 0.0001). Immunostaining of endogrin, a marker of endothelial proliferation, increased in the microvessels located in the fibrotic interstitial spaces. These results suggest that angiogenesis may play a critical role in the progression of tubulointerstitial injuries and that up-regulation of PD-ECGF may contribute to neovascularization.     

Open Wound Healing In Vivo: Monitoring Binding and Presence of Adhesion/Growth-Regulatory Galectins in Rat Skin during the Course of Complete Re-Epithelialization

Galectins are a family of carbohydrate-binding proteins that modulate inflammation and immunity. This functional versatility prompted us to perform a histochemical study of their occurrence during wound healing using rat skin as an in vivo model. Wound healing is a dynamic process that exhibits three basic phases: inflammation, proliferation, and maturation. In this study antibodies against keratins-10 and -14, wide-spectrum cytokeratin, vimentin, and fibronectin, and non-cross-reactive antibodies to galectins-1, -2, and -3 were applied to frozen sections of skin specimens two days (inflammatory phase), seven days (proliferation phase), and twenty-one days (maturation phase) after wounding. The presence of binding sites for galectins-1, -2, -3, and -7 as a measure for assessing changes in reactivity was determined using labeled proteins as probes. Our study detected a series of alterations in galectin parameters during the different phases of wound healing. Presence of galectin-1, for example, increased during the early phase of healing, whereas galectin-3 rapidly decreased in newly formed granulation tissue. In addition, nuclear reactivity of epidermal cells for galectin-2 occurred seven days post-trauma. The dynamic regulation of galectins during re-epithelialization intimates a role of these proteins in skin wound healing, most notably for galectin-1 increasing during the early phases and galectin-3 then slightly increasing during later phases of healing. Such changes may identify a potential target for the development of novel drugs to aid in wound repair and patients' care.     

Proliferative index in breast carcinoma determined in situ by Ki67 immunostaining and its relationship to clinical and pathological variables

Sixty cases of primary breast carcinoma have been studied using a monoclonal antibody, Ki67, which recognizes an antigen expressed by cells in G1, S, G2, and M phases of the cell cycle but not Go. A Ki67 score (positive cells/total tumour cells) was determined, and possible relationships between this index of cellular proliferation and a number of clinical and pathological parameters were investigated. There was a strong positive correlation between the Ki67 score and mitotic index (p less than 0.001), a weak negative correlation with age (p less than 0.02), and weak positive correlations with histological tumour grade (p less than 0.03), tumour necrosis (p less than 0.01), and cellular reaction (p less than 0.01). No relationship was noted between the Ki67 score and tumour size, nodal status, tumour oestrogen receptor levels, or menopausal status. The Ki67 score may prove to be an objective indicator of biological behaviour and thus be of clinical significance, particularly since it is not strongly related to other clinical and pathological parameters used in predicting outcome in breast carcinoma.     

Proliferative potential and DNA repair in lymphocytes from short-lived and long-lived strains of mice, relation to aging

The replicative capacity and DNA repair ability of spleen lymphocytes of young and old mice from short-lived and long-lived strains were studied. DNA repair after both UV- and gamma-induced damage was investigated. Proliferation after Con A decreased as a function of age in both mouse strains and paralleled an age-associated decline in repair of DNA damage induced by either UV or gamma-irradiation. Compared to the long-lived, the short-lived strain displayed an earlier impairment of both proliferative and repair potentials. DNA repair after gamma-induced damage only occurred if lymphocytes were stimulated to proliferate. Resting lymphocytes appeared unable to repair strand breaks. By contrast, DNA repair of UV-induced damage showed two components: one was dependent on the cell proliferative state, the other was not. Both components were stimulated or induced by mitogen. Resting lymphocytes were able to perform an appreciable amount of repair after UV irradiation. Our results suggest that resting or post-mitotic cells possess a greater possibility to regulate repair of UV-induced than gamma-induced damage. We speculate that it may be the level of this proliferation-independent, but mitogen inducible form of repair which correlates with maximum life-spans between species, thereby explaining why repair of UV- but not gamma-induced damage reveals such a correlation.     

Perlecan Knockdown in Metastatic Prostate Cancer Cells Reduces Heparin-binding Growth Factor Responses in vitro and Tumor Growth in vivo

Perlecan (Pln) is a major heparan sulfate proteoglycan (HSPG) of extracellular matrices and bone marrow stroma. Pln, via glycosaminoglycans in domains I and V, acts as a co-receptor for delivery of heparin binding growth factors (HBGFs) that support cancer growth and vascularization. Specifically, glycosaminoglycans bind HBGFs and activate HBGF receptors, including those for FGF-2 and VEGF-A. The contribution of Pln to prostate cancer growth was tested using a ribozyme approach to knockdown Pln expression levels. Transfection into the androgen-independent, bone targeted prostate cancer line, C4-2B, and efficient stable knockdown of Pln was demonstrated by quantitative PCR, immunohistochemistry and immunoblotting. Three individually isolated subclones with 75–80% knockdown in Pln mRNA, protein expression and secretion into ECM were used to study in vitro growth responses to FGF-2 and VEGF-A. While cells with normal Pln levels responded to both HBGFs, knockdown cells responded poorly. All lines responded to serum growth factors and IGF-I. Anchorage-independent growth assays showed reduced colony size and cohesiveness by all Pln deficient subclones compared to parental C4-2B cells. In vivo effects of Pln knockdown were measured by inoculating knockdown and control ribozyme transfected cell lines into athymic mice. A reduced growth rate, smaller tumor size, diminished vascularization and failure to elevate serum PSA characterized mice bearing Pln knockdown C4-2B cells. Poor vascularization correlated with reduced levels of VEGF-A secreted by Pln knockdown lines. We conclude that Pln is an essential ECM component involved in growth responses of metastatic prostate cancer cells to HBGFs deposited in local and metastatic microenvironment.