Faculty Bibliography

OBJECTIVE: To investigate the presence and cellular distribution of transforming growth factor (TGF)-beta s in surgically induced pelvic fibrous adhesions in rat uterine horns subjected to burn, crush, and debridement injury. METHODS: Thirty injured and 20 uninjured rats were treated postoperatively with intraperitoneal administration of either 2 micrograms/mL of recombinant human TGF-beta, 10 micrograms/mL TGF-beta neutralizing antibody, or phosphate-buffered saline + 500 micrograms rat serum albumin for 5 consecutive days. The intact (uninjured) and fibrous tissues were analyzed immunohistochemically for the presence of TGF-beta s using polyclonal antibodies to TGF-beta s 1-3. RESULTS: The intact peritoneum immunostained with a lower intensity than fibrous adhesive tissues for TGF-beta 1, TGF-beta 2, and TGF-beta 3. The immunoreactive TGF-beta s were present in fibroblasts, inflammatory cells infiltrated into the fibrous adhesion, and endothelial and smooth-muscle cells of the arterioles. In the uterine tissue at the site of injury, the following immunostained for TGF-beta s: uterine serosal tissue, myometrial smooth-muscle cells, endometrial luminal and glandular epithelial cells, and inflammatory cells. However, endometrial stromal cells did not immunostain for TGF-beta s. There were no substantial differences in immunostaining intensities of fibrous adhesive tissues in the TGF-beta group, neutralizing TGF-beta antibody group, and the controls. CONCLUSION: The data suggest that TGF-beta s may play a role in the formation and maintenance of fibrous adhesions following intraperitoneal injury

Active exogenous transforming growth factor-beta s (TGF-beta s) are potent modulators of extracellular matrix synthesis in cell culture and stimulate matrix synthesis in wounds and other remodeling tissues. The role of endogenous TGF-beta s in remodeling tissues is less well defined. Vascular remodeling in the pulmonary arteries of patients with primary pulmonary hypertension is characterized, in part, by abnormal deposition of immunohistochemically detectable procollagen, thereby identifying actively remodeling vessels. We used this marker of active matrix synthesis to begin defining the in vivo role of TGF-beta in the complex milieu of actively remodeling tissues. Immunohistochemistry using isoform-specific anti-TGF-beta antibodies was performed to determine whether TGF-beta was present in actively remodeling hypertensive pulmonary arteries 20 to 500 microns in diameter. Intense, cell-associated TGF-beta 3 immunoreactivity was observed in the media and neointima of these hypertensive muscular arteries. Immunostaining was present, but less intense, in normal arteries of comparable size. TGF-beta 2 immunoreactivity was observed in normal vessels and was increased slightly in hypertensive vessels, in a pattern resembling TGF-beta 3 immunoreactivity. No staining was associated with the adventitia. TGF-beta 1 immunostaining was either faint or absent in both normal and hypertensive vessels. Comparison of procollagen and TGF-beta localization demonstrated that TGF-beta 2 and TGF-beta 3 colocalized at all sites of procollagen synthesis. However, TGF-beta was observed in vessels, or vascular compartments, where there was no procollagen synthesis. Procollagen immunoreactivity was not present in normal vessels that showed immunoreactivity for TGF-beta 2 and TGF-beta 3. These observations suggest: a) the stimulation of procollagen synthesis by TGF-beta in vivo is more complex than suggested by in vitro studies and b) a potential role for TGF-beta 2 or TGF-beta 3, but not TGF-beta 1, in hypertensive pulmonary vascular remodeling

Historically, the role for transforming growth factor-Ps (TGF-beta-s) in malignancy has been difficult to prove. TGF-beta-s are potent autocrine and paracrine negative regulators of the growth of normal epithelial and neuroectodermal cells in vitro. Therefore we hypothesized that the aberrant growth of tumor cells and neoplastic development, in general, may be due to a loss of growth regulation by TGF-beta. By immunohistochemistry, using isoform specific antibodies raised to synthetic peptides of each TGF-beta isoform, and by in situ hybridization, using non-cross-hybridizing cRNA probes to each TGF-beta isoform, we examined the protein and mRNA expression of TGF-beta, respectively, in a variety of carcinomas and gliomas compared to normal tissue. The analysis of the cell types that synthesized the message for TGF-beta isoforms compared to those which synthesized the protein indicated whether TGF-beta functioned by an autocrine or paracrine mechanism of action in these tumors

The transforming growth factors beta (TGFs-beta) are potent inhibitors of cell proliferation and are usually secreted in a latent form. TGF-beta 1, TGF-beta 2, and TGF-beta 3 are expressed in distinct but overlapping patterns in the developing mouse mammary gland. To study the role of transforming growth factor-beta 1 (TGF-beta 1) in normal mammary development and in mammary neoplasia, we have constructed three transgenic mouse lines that express a simian TGF-beta 1 s223/225 mutated to produce a constitutively active product under the control of the MMTV enhancer/promoter. Expression of the transgene, as confirmed by in situ hybridization, immunohistochemistry, and Northern blot analysis, was associated with marked suppression of the normal pattern of mammary ductal tree development in female transgenics. Reduction in total ductal tree volume was observed at 7 weeks, soon after estrous begins, and was most apparent at 13 weeks, as ductal growth in the normal mammary gland declines. This effect was seen in all three lines. However, during pregnancy, alveolar outgrowths developed from the hypoplastic ductal tree, and lactation occurred, therefore, all transgenic females could feed full litters. Unlike many other transgenic mouse models in which expression of growth factors or oncogenes under control of the MMTV promoter leads to mammary epithelial hyperplasia and increased tumor formation, the MMTV-TGF-beta 1S223/225 transgene causes conditional hypoplasia of the mammary ductal tree and no spontaneous tumors have been detected in the MMTV-TGF-beta 1S223/225 transgenic animals

BACKGROUND: Transforming growth factor beta s (TGF-beta s) constitute a family of bifunctional polypeptide growth factors that either inhibit or stimulate cell proliferation. Perturbations in TGF-beta expression and function may lead to loss of negative constraints on cell growth. In this study, we examined TGF-beta expression in human pancreatic cancer. METHODS: The distribution of TGF-beta isoforms in 60 human pancreatic cancers was examined using immunohistochemical, Northern blot, and in situ hybridization techniques. RESULTS: Immunohistochemical analysis showed the presence of TGF-beta 1 (47% of tumors), TGF-beta 2 (42% of tumors), and TGF-beta 3 (40% of tumors) in the cancer cells. The presence of TGF-beta 2 was associated with advanced tumor stage (P < 0.05). Furthermore, there was a significant correlation between the absence of TGF-beta s in the tumors and longer postoperative survival. Northern blot analysis indicated that, by comparison with the normal pancreas, pancreatic adenocarcinomas showed 11- (P < 0.001), 7- (P < 0.05), and 9-fold (P < 0.001) increases in the messenger RNA (mRNA) levels encoding TGF-beta 1, TGF-beta 2, and TGF-beta 3, respectively. By in situ hybridization, these mRNA moieties colocalized with their respective proteins in the cancer cells. CONCLUSIONS: These findings show that human pancreatic cancers show increased levels of TGF-beta isoforms and enhanced TGF-beta mRNA expression and suggest that the presence of TGF-beta s in pancreatic cancer cells may contribute to disease progression

The arterial wall responds to thrombosis or mechanical injury through the induction of specific gene products that increase cellular proliferation and connective tissue formation. These changes result in intimal hyperplasia that is observed in restenosis and the early phases of atherosclerosis. Transforming growth factor beta 1 (TGF-beta 1) is a secreted multi-functional protein that plays an important role in embryonal development and in repair following tissue injury. However, the function of TGF-beta 1 in vascular cell growth in vivo has not been defined. In this report, we have evaluated the role of TGF-beta 1 in the pathophysiology of intimal and medial hyperplasia by gene transfer of an expression plasmid encoding active TGF-beta 1 into porcine arteries. Expression of TGF-beta 1 in normal arteries resulted in substantial extracellular matrix production accompanied by intimal and medial hyperplasia. Increased procollagen, collagen, and proteoglycan synthesis in the neointima was demonstrated by immunohistochemistry relative to control transfected arteries. Expression of TGF-beta 1 induced a distinctly different program of gene expression and biologic response from the platelet-derived growth factor B (PDGF B) gene: procollagen synthesis induced by TGF-beta 1 was greater, and cellular proliferation was less prominent. These findings show that TGF-beta 1 differentially modulates extracellular matrix production and cellular proliferation in the arterial wall in vivo and could play a reparative role in the response to arterial injury.

'Classical' chemoattractants, such as FMLP, C5a, or leukotriene B4, not only elicit directed motility but also activate neutrophils (degranulation, release of active oxygen species). Signal transduction after ligation of receptors for these classical chemoattractants is mediated by pertussis toxin (PT)-sensitive, heterotrimeric G proteins and the early production of lipid messengers via phospholipases. In contrast, we have previously shown that substance P (SP) and transforming growth factor-beta 1 (TGF-beta 1) are 'pure' chemoattractants in that they elicit chemotaxis without activating neutrophils. Paradoxically, pure chemoattractants also activate G proteins (plasmalemmal GTPase activity) without eliciting increments in cytosolic calcium ([Ca]i) and thus inositol trisphosphate. We therefore determined lipid remodeling and signal transduction in response to pure chemoattractants. Increments in plasmalemmal GTPase activated by SP (0.1 microM) and TGF-beta 1 (40 fM), like that after FMLP, were PT-sensitive (SP = 6.6 +/- 2 pm/mg/min vs SP + PT = 1.1 +/- 0.9 over basal activity; TGF-beta 1 = 4.3 +/- 1.6 vs TGF-beta 1 + PT = 2.3 +/- 0.9). In parallel, treatment of PMN with PT (1 microgram/ml, 30 min) inhibited chemotaxis (under agarose) after FMLP (2175 +/- 176 (SEM) microns vs 726 +/- 267) and SP (411 +/- 99 microns vs 103 +/- 62 microns) and TGF-beta 1 (40 fM, 375 +/- 53 microns vs 83 +/- 47). However, G proteins coupled to receptors for SP and TGF-beta 1, unlike FMLP, did not appear to be linked to phospholipases in that neither increments in diacylglycerol were detected after receptor ligation (FMLP = 152 +/- 22% resting levels; SP = 101 +/- 5%; TGF-beta 1 = 105 +/- 4%) nor was alkylacylglycerol increased by exposure to SP or TGF-beta 1 (SP = 92 +/- 4%; TGF-beta 1 = 101 +/- 8%; FMLP = 226 +/- 40%). Moreover, polymorphonuclear leukocytes failed to generate phosphatidates (PA) of either species after SP (DA-PA = 79 +/- 9% resting at 60 s; EA-PA = 103 +/- 4%) or TGF-beta 1 (DA-PA = 101 +/- 5%; EA-PA = 98 +/- 9%) in contrast to FMLP (DA-PA = 155 +/- 22%; EA-PA = 149 +/- 16%). The data clearly contravene the current dogma that all chemoattractants use inositol trisphosphate and diglycerides as intracellular signals and suggest the presence of a unique subset of PT-sensitive G proteins, not coupled to 'classical' phospholipases, transduce chemoattraction

Transforming growth factor beta (TGF-beta) regulates cellular growth and differentiation and stimulates the synthesis and secretion of protein constituents of the extracellular matrix. Three isoforms of TGF-beta have been found in mammals. Although the biological activities of TGF-beta 1, TGF-beta 2, and TGF-beta 3 are similar at the level of cell culture, distinct in vivo functions for these molecules are emerging. To gain insight into the role of each isoform in wound repair, antibodies specific for each isoform of TGF-beta were used to examine excisional wound repair. Marked differences in the temporal and spatial relationships for immunoreactive TGF-beta 1, -beta 2, and -beta 3 were noted throughout the repair process. TGF-beta 2 and TGF-beta 3 were prevalent by 24 hours after excisional wounding, and strong immunoreactivity was observed in the migrating epidermis. Subtle changes in immunoreactivity occurred for TGF-beta 2 and TGF-beta 3 in cells of the epidermal appendages, mesenchymal derivatives, granulation tissue, and the underlying dermis throughout wound repair. In contrast, TGF-beta 1 was not associated with any undifferentiated cells and was not present in the dermis and most dermal structures in both nonwounded skin or wounds until day 5 after wounding, when re-epithelialization was complete. Following re-epithelialization, TGF-beta 2 and TGF-beta 3 were present in all four layers of stratum corneum of the differentiating epidermis. All three TGF-beta isoforms were present in mesenchymal cells and basal lamina, suggesting their role in the modulation of dermal-epidermal interaction during wound repair. Our observations support individual in vivo function for TGF-beta isoforms in cutaneous wound repair

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease in which peripheral lymphoid cells are activated by immunization with myelin proteins and become effector cells that traverse the central nervous system (CNS) capillaries and initiate inflammatory demyelinating lesions. The administration of transforming growth factor-beta (TGF-beta) has been shown previously to decrease the incidence and severity of EAE. In our studies we have determined: 1) the effects of TGF-beta injected at different intervals after the EAE-inducing immunization; 2) the effect of TGF-beta on the development of sensitized T cells, as assayed by the proliferative responses of T cells from lymph nodes and peripheral blood; 3) the extent of lymphoid cell infiltration in CNS of TGF-beta-treated and control mice; and 4) the role of endogenous TGF-beta and TNF in determining the severity of both acute and relapsing EAE. The onset of acute-EAE in SJL mice, induced by immunization with spinal cord homogenate in CFA and pertussigen, is on days 10 to 15. Although daily i.p. injections of 0.2 to 2 micrograms TGF-beta 1 or TGF-beta 2 on days 5 to 9 after immunization are highly protective, injections on days 1 to 5 or 9 to 13 are not. Moreover, anti-TGF-beta accelerates and aggrevates EAE when given on days 5 and 9, but not on day 12. Anti-TNF, injected on days 5 and 9, provides a comparable degree of protection as does TGF-beta. Similarly, in relapsing EAE, anti-TGF-beta increases, whereas anti-TNF decreases the incidence and severity of relapses. TGF-beta treatment on days 5 to 9 does not influence the appearance of sensitized cells in peripheral blood and lymph nodes, but does prevent the accumulation of T cells in brain and spinal cord, as assayed on days 15 to 20. It is concluded that the protective effect of TGF-beta is exerted at the level of the target organ, CNS and/or its vascular endothelium, rather than through a direct effect on lymphoid cells, and that there is a small window of 4 days in which TGF-beta exerts its protective effect