Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Hyaluronan production in human rheumatoid fibroblastic synovial lining cells is increased by interleukin 1β but inhibited by transforming growth factor β1

OBJECTIVES—To investigate the regulatory roles of interleukin 1β (IL1β), tumour necrosis factor α (TNFα), interferon γ (IFNγ) or transforming growth factor β1 (TGFβ1) on hyaluronan (HA) synthesis by human fibroblastic synovial lining cells.
METHODS—Concentrations of HA in culture supernatants of fibroblastic synovial lining cell line (RAMAK-1 cell line) with or without stimulation by IL1β, TNFα, IFNγ or TGFβ1 were measured by sandwich binding protein assay. Levels of HA synthase mRNA of the cells with or without stimulation were detected by reverse transcribed polymerase chain reaction. Molecular weights of HA in the culture supernatants of the cells with or without stimulation were measured using high performance gel permeation liquid chromatography.
RESULTS—HA synthesis by the cells was not significantly augmented by TNFα or by IFNγ. It was significantly stimulated by IL1β but inhibited by TGFβ1. Molecular weights of HA in the culture supernatants of the cells were unchanged by stimulation with TNFα. They were remarkably increased by stimulation with IL1β and IFNγ, but reduced with TGFβ1.
CONCLUSION—IL1β is an up regulator of HA synthesis, while TGFβ1 is a down regulator. HA production in the synovial lining cells of inflamed joints (for example, rheumatoid arthritis) might be regulated by the balance of these cytokines.

Keywords: synovial lining cells; hyaluronan, interleukin 1β; transforming growth factor β1     

Evidence for pro-β-nerve growth factor, a biosynthetic precursor to β-nerve growth factor

The biosynthesis of beta-nerve growth factor (betaNGF) was studied in mouse submaxillary glands incubated with L-[(35)S]cystine. betaNGF was isolated from tissue extracts by the addition of antiserum against betaNGF and the washed immunoprecipitates were analyzed by sodium dodecyl sulfate gel electrophoresis. With short labeling periods (10 and 25 min) there is a major labeled species with an apparent molecular weight of 22,000 and a smaller peak comigrating with purified betaNGF chains (13,260). As time proceeds, the radioactivity in the 22,000 molecular weight peak plateaus, while the label in betaNGF continues to increase, until by 4 hr it greatly exceeds the radioactivity of the 22,000 molecular weight species. When glands incubated for 10 min are transferred to medium containing a large excess of unlabeled L-cystine, the 22,000 molecular weight peak gradually declines, and there is a corresponding increase in radioactivity at the betaNGF position. The 22,000 molecular weight species isolated from sodium dodecyl sulfate gels possesses all the cystine-containing peptides of betaNGF, and possibly two additional ones. When immunoprecipitates from submaxillary glands labeled for 25 min are incubated with the gamma subunit (a specific arginyl-esteropeptidase associated with betaNGF in the 7S NGF complex), the radioactivity in the 22,000 molecular weight species is converted to the betaNGF position. The results suggest that the 22,000 molecular weight species is a biosynthetic precursor to betaNGF, and that the gamma subunit may function as a specific protease in the processing event.     

Morphologic and mitogenic responses of rabbit synovial fibroblasts to transforming growth factor β require transforming growth factor α or epidermal growth factor

We tested the hypothesis that normal synovial fibroblasts might proliferate in response to transforming growth factors (TGFs), peptides that are extracted with acid-ethanol from bovine kidney or salivary gland and that cause anchorage-independent growth of normal cells. A 72-hour exposure of confluent monolayers of rabbit synovial fibroblasts in 10% fetal calf serum to partially purified TGF-β in the presence of TGF-β gave a 2- to 5-fold increase in incorporation of ³H-thymidine, protein content, and cell number. Similar results were obtained with high pressure liquid chromatography-purified TGF-β in the presence of epidermal growth factor (EGF) (a type of TGF-β). By itself, purified TGF-β was not mitogenic; it depended absolutely on EGF. However, only TGF-β along with EGF, and not EGF alone, induced a marked morphologic change: a piling up of cells into foci resembling those commonly seen in primary cultures of rheumatoid synovial cells. Mitogenic responses induced by the TGF-β-EGF combination were prevented by all-trans-retinoic acid but not by indomethacin or dexamethasone. The data indicate that TGF-β, a peptide extracted from normal cells, can act in concert with EGF to cause proliferation and piling up of synovial cells and raise the possibility that these factors may play a role in rheumatoid arthritis and other proliferative but nonmalignant diseases as well.     

Increased expression of transforming growth factor α precursors in acute experimental colitis in rats

Background and aim—Epidermal growth factor (EGF)and transforming growth factor α (TGF-α), members of the EGF familyof growth factors, protect rat gastric and colonic mucosa againstinjury. Having shown previously that exogenously applied EGF protects rat colonic mucosa against injury, the aim of the present study was toevaluate the endogenously expressed ligand mediating the protectiveeffect of EGF/TGF-α in vivo.
Methods—In an experimental model oftrinitrobenzene sulphonic acid (TNBS)/ ethanol induced colitis in ratsEGF and TGF-α expression was evaluated using a ribonucleaseprotection assay, northern blot analysis, western blot analysis, and immunohistochemistry.
Results—TGF-α mRNA increased 3-4 times at 4-8hours after induction of colitis and returned to control levels within24 hours. TGF-α immunoreactive protein with a molecular size of about28kDa representing TGF-α precursors increased markedly afterinduction of colitis with a peak at 8-12 hours. No fully processed 5.6 kDa TGF-α protein was detected in normal or inflamed colon tissue. Only a weak signal for EGF mRNA expression was detected in the ratcolon and no EGF protein was observed by immunohistochemistry orwestern blot analysis.
Conclusions—TGF-α precursors are the mainligands for the EGF receptor in acute colitis. It is hypothesised thatTGF-α precursors convey the biological activity of endogenous TGF-αpeptides during mucosal defence and repair.

     

Loss of functional cell surface transforming growth factor β (TGF-β) type 1 receptor correlates with insensitivity to TGF-β in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries, and there is significant variability in survival within CLL clinical stages. Earlier studies showed that CLL cells produce and are usually growth inhibited by transforming growth factor β type 1 (TGF-β1), suggesting a mechanism for the clinically indolent course of most CLL. Here we studied the mechanism by which CLL cells from about one-third of the patients are insensitive to TGF-β1. Of the 13 patients studied, CLL cells isolated from the peripheral blood of 8 patients were sensitive to growth inhibition by TGF-β1, as determined by incorporation of tritiated thymidine, whereas those from 5 patients were completely resistant to TGF-β1. As judged by binding of radiolabeled TGF-β1 followed by cross-linking and immunoprecipitation with anti-receptor antisera, CLL cells sensitive to TGF-β1 exhibited normal cell surface expression of both types 1 and 2 TGF-β receptors. In contrast, all CLL cells resistant to TGF-β1 exhibited no detectable surface type I receptors able to bind TGF-β1, but normal expression of type II receptors. Both TGF-β1-sensitive and TGF-β1-resistant CLL cells contained normal amounts of both type 1 and type 2 receptor mRNAs. Specific loss of type 1 receptor expression represents a new mechanism by which cells acquire resistance to TGF-β1-mediated growth inhibition in the development and progression of human lymphoproliferative malignancies.     

Consistent loss of functional transforming growth factor β receptor expression in murine plasmacytomas

Murine plasmacytomas are tumors of Ig-secreting plasma cells that can be induced in genetically susceptible BALB/c mice. The deregulation of the c-myc protooncogene is a critical oncogenic event in the development of plasmacytomas (PCTs) although it is not sufficient for their malignant transformation. We have demonstrated that PCTs produce active transforming growth factor β (TGF-β) in vitro. Because TGF-β is a potent negative regulator of the proliferation and differentiation of B lymphocytes, we examined its role in plasmacytomagenesis by comparing responsiveness to TGF-β of nonneoplastic plasma cells and PCTs. The nontransformed plasma cells that accumulate in interleukin 6 transgenic mice undergo accelerated apoptosis upon treatment with TGF-β, but the 15 PCTs studied, including primary and transplanted tumors as well as established cell lines, were refractory to TGF-β-mediated growth inhibition and apoptosis. Although PCTs lack functional TGF-β receptors as demonstrated by chemical crosslinking to radiolabeled TGF-β1, they nonetheless contain mRNA and protein for both type I and II TGF-β receptors, suggesting a potential defect in receptor trafficking or processing. The results clearly show the consistent inactivation of TGF-β receptors in plasmacytoma cells, demonstrating for the first time that interruption of a tumor suppressor pathway contributes to plasmacytomagenesis.     

Involvement of αvβ5 integrin–mediated activation of latent transforming growth factor β1 in autocrine transforming growth factor β signaling in systemic sclerosis fibroblasts

Objective

To confirm the involvement of αvβ5 in the self‐activation system in systemic sclerosis (SSc) fibroblasts.

Methods

Levels of αvβ5 expression were analyzed by immunoprecipitation. The promoter activity of the human α2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor β (TGFβ) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.

Results

Levels of αvβ5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti‐αvβ5 antibody or β5 antisense oligonucleotide significantly reduced human α2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFβ1 antisense oligonucleotide, the exogenous latent TGFβ1 stimulation significantly increased human α2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti‐αvβ5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with β5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti‐αvβ5 antibody. Anti‐αvβ5 antibody reversed the myofibroblastic features of SSc fibroblasts.

Conclusion

Up‐regulated expression of αvβ5 contributes to the establishment of autocrine TGFβ signaling in SSc fibroblasts through activation of endogenous latent TGFβ1.

     

Fibroblast growth factor 2 and transforming growth factor β1 induce precocious maturation of articular cartilage

Objective

We have discovered that a combination of fibroblast growth factor 2 and transforming growth factor β1 induce profound morphologic changes in immature articular cartilage. The purpose of this study was to test the hypothesis that these changes represent accelerated postnatal maturation.

Methods

Histochemical and biochemical assays were used to confirm the nature of the morphologic changes that accompany growth factor stimulation of immature bovine articular cartilage explants in serum‐free culture medium. Growth factor–induced apoptosis, cellular proliferation, and changes in the collagen network were also quantitatively analyzed.

Results

Growth factor stimulation resulted in rapid resorption from the basal aspect of immature cartilage explants that was simultaneously opposed by cellular proliferation from the apical aspect driven from a pool of chondroprogenitor cells we have previously described. Maturation‐dependent changes in tissue stiffness, collagen crosslinking, and collagen fibril architecture as well as differentiation of the extracellular matrix into distinct pericellular, territorial, and interterritorial domains were all present in growth factor–stimulated cartilage samples and absent in control samples.

Conclusion

Our data demonstrate that it is possible to significantly enhance the maturation of cartilage tissue using specific growth factor stimulation. This may have applications in transplantation therapy or in the treatment of diseased cartilage, through phenotype modulation of osteoarthritic chondrocytes in order to stimulate growth and maturation of cartilage repair tissue.

     

Inhibition of fas antigen-mediated apoptosis of rheumatoid synovial cells in vitro by transforming growth factor β1

Objective. To investigate the mitogenic and anti-apoptotic effects of transforming growth factor β1 (TGFβ) on rheumatoid synovial cells in vitro. Methods. Synovial cells were cultured with or without TGFβ1. After incubation, the proliferative response of synovial cells and the expression of Fas antigen and bcl-2 on synovial cells were examined. Finally, Fas antigen-mediated apoptosis of synovial cells was investigated by the addition of anti-Fas antibody. Results. TGFβ1 enhanced the proliferation of synovial cells in a dose-dependent manner. In addition, Fas antigen expression on synovial cells was inhibited by the addition of TGFβ1 with up-regulation of bcl-2 expression. The addition of anti-Fas antibody induced synovial cell apoptosis. However, stimulation of synovial cells with TGFβ1 became markedly resistant to Fas antigen-mediated apoptosis. The results were not affected by the addition of a neutralizing antibody to platelet-derived growth factor type AA (PDGF-AA), which suggests that the effect of TGFβ1 on synovial cells was promoted via PDGF-AA-independent mechanisms. Conclusion. Our results suggest that TGFβ1 promotes synovial cell proliferation through its mitogenic effect on synovial cells and interference with the apoptotic process mediated by the Fas antigen, resulting in the perpetuation of the synovial hyperplasia in patients with rheumatoid arthritis.     

Reduced susceptibility of mice overexpressing transforming growth factor α to dextran sodium sulphate induced colitis

Background—Transforminggrowth factor α (TGF-α) knockout mice have increased susceptibilityto dextran sodium sulphate (DSS) induced colitis.
Aim—To substantiatethe findings that TGF-α is a key mediator of colonic mucosalprotection and/or repair mechanisms by evaluating the susceptibility ofmice overexpressing TGF-α to DSS induced colitis.
Methods—TGF-αoverexpression was induced in transgenic mice by ZnSO₄administration in drinking water (TG+). Three groups were used ascontrols: one transgenic group without ZnSO₄ administration (TG−), and two non-transgenic littermate groups receivingZnSO₄ (Non-TG+) or only water (Non-TG−). Acute colitiswas induced in all groups by administration of DSS (5%, w/v) indrinking water for six days ad libitum.
Results—About 35-39%of the entire colonic mucosa was destroyed in Non-TG−, Non-TG+, andTG− animals compared with 9% in TG+ mice. The crypt damage score was18.7 (0.9), 18.2 (1.0), 18.9(0.8), and 6.8 (1.5) (means (SEM)) inNon-TG−, Non-TG+, TG−, and TG+ mice respectively. Mucin andbromodeoxyuridine staining were markedly enhanced in colons of TG+ micecompared with controls, indicating increased mucosal protection and regeneration.
Conclusions—Thesignificantly reduced susceptibility of mice overexpressing TGF-α toDSS further substantiates that endogenous TGF-α is a pivotal mediatorof protection and/or healing mechanisms in the colon.

Keywords:transforming growth factor α; epidermal growthfactor; dextran sodium sulphate; colitis; inflammatory bowel disease; transgenic mice

     

Human eosinophils regulate human lung- and skin-derived fibroblast properties in vitro: A role for transforming growth factor β (TGF-β)

Eosinophils have been associated with fibrosis. To investigate their direct role in fibrosis, human peripheral blood eosinophil sonicate was added to human lung or dermal fibroblasts, and proliferation ([(3)H]thymidine) and collagen synthesis ([(3)H]proline) were evaluated. Proliferation was enhanced significantly in the monolayers in a dose-dependent manner. The activity of the eosinophil fibrogenic factor(s) remained unaltered when heated (56 degrees C, 30 min). Supernatants of cultured eosinophils (20 min or 18 hr) also enhanced lung fibroblast proliferation, indicating that the preformed mitogenic factor(s) can be released both promptly and with a long kinetic. Eosinophils significantly decreased collagen production in lung fibroblasts while increasing it in dermal fibroblasts. However, eosinophils containing matrix metalloproteinase 9 (zymography) in latent form and tissue inhibitors of metalloproteinases 1 and 2 (reverse zymography) did not influence either fibroblast matrix metalloproteinases or tissue inhibitors of metalloproteinases. Eosinophil sonicate added to skin and lung fibroblasts in tridimensional collagen lattices significantly enhanced lattice contraction. Transforming growth factor beta (TGF-beta) is a major fibrogenic cytokine produced by eosinophils. Therefore, to assess its role, eosinophil sonicate was preincubated with anti-TGF-beta neutralizing antibodies. This treatment partially inhibited proliferation of lung and collagen synthesis of dermal fibroblasts and suppressed the stimulation of lattice contraction, indicating the fibrogenic role of eosinophil-associated TGF-beta. In conclusion, we have shown that eosinophils act as direct modulatory cells in fibroblast proliferation, collagen synthesis, and lattice contraction, in part, through TGF-beta. These data corroborate the importance of eosinophils in skin and lung fibrosis.     

Expression of a dominant-negative type II transforming growth factor β (TGF-β) receptor in the epidermis of transgenic mice blocks TGF-β-mediated growth inhibition

To determine whether a functional type II receptor of transforming growth factor β (TGF-β) is required to mediate the growth inhibitory effect of TGF-β on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-β receptor (ΔβRII) in the epidermis. The ΔβRII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10–14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F₁ mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from ΔβRII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-β-induced growth inhibition. These data document the role of the type II TGF-β receptor in mediating TGF-β-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.     

Dysregulation of transforming growth factor β signaling in scleroderma: Overexpression of endoglin in cutaneous scleroderma fibroblasts

Objective

As an initial approach to understanding the basis of the systemic sclerosis (SSc; scleroderma) phenotype, we sought to identify genes in the transforming growth factor β (TGFβ) signaling pathway that are up‐regulated in lesional SSc fibroblasts relative to their normal counterparts.

Methods

We used gene chip, differential display, fluorescence‐activated cell sorter, and overexpression analyses to assess the potential role of TGFβ signaling components in fibrosis. Fibroblasts were obtained by punch biopsy from patients with diffuse cutaneous SSc of 2–14 months' duration (mean 8 months) and from age‐ and sex‐matched healthy control subjects.

Results

Unexpectedly, we found that fibroblasts from SSc patients showed elevated expression of the endothelial cell–enriched TGFβ receptor endoglin. Endoglin is a member of the nonsignaling high‐affinity TGFβ receptor type III family. The expression of endoglin increased with progression of disease. Transfection of endoglin in fibroblasts suppressed the TGFβ‐mediated induction of connective tissue growth factor promoter activity.

Conclusion

SSc is characterized by overproduction of matrix; that is, genes that are targets of TGFβ signaling in normal fibroblasts. Our findings suggest that lesional SSc fibroblasts may overexpress endoglin as a negative feedback mechanism in an attempt to block further induction of profibrotic genes by TGFβ.

     

Pancreatic β cells synthesize and secrete nerve growth factor

Differentiation and function of pancreatic β cells are regulated by a variety of hormones and growth factors, including nerve growth factor (NGF). Whether this is an endocrine or autocrine/paracrine role for NGF is not known. We demonstrate that NGF is produced and secreted by adult rat pancreatic β cells. NGF secretion is increased in response to elevated glucose or potassium, but decreased in response to dibutyryl cAMP. Moreover, steady-state levels of NGF mRNA are down-regulated by dibutyryl cAMP, which is opposite to the effect of cAMP on insulin release. NGF-stimulated changes in morphology and function are mediated by high-affinity Trk A receptors in other mammalian cells. Trk A receptors are present in β cells and steady-state levels of Trk A mRNA are modulated by NGF and dibutyryl cAMP. Taken together, these findings suggest endocrine and autocrine roles for pancreatic β-cell NGF, which, in turn, could be related to the pathogenesis of diabetes mellitus where serum NGF levels are diminished.