Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Here we define a population of resident lung mesenchymal stem cells (luMSC) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin induced loss of these endogenous luMSC and elicited fibrosis (PF), inflammation and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSC attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T cell proliferation in vitro. Global gene expression analysis indicated that the luMSC are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and pro-fibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity following injury however when endogenous MSC are lost this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury.

Alveolar and lung liquid clearances were studied over 1, 4, and 6 h in intact anesthetized ventilated rats by instillation of 5% albumin solution with 1.5 microCi of 125I-labeled albumin (3 ml/kg into 1 lung or 6 ml/kg into both lungs). Alveolar protein clearance as measured by residual 125I-albumin in the lung over 6 h was similar to the slow rates measured in other species. Alveolar liquid clearance was estimated by the concentration of albumin in the air spaces. After 1 h, this concentration was 7.8 +/- 0.7 g/dl, which was significantly greater than the initial protein concentration of 5.3 +/- 0.2 g/dl (P < 0.05). Amiloride (10(-3) M) inhibited 45% of the basal alveolar liquid clearance, and ouabain (10(-3) M), instilled and intravenously infused (0.004 mg), inhibited 30% of the clearance. beta-Adrenergic agonist instillation increased alveolar liquid clearance to the fastest 1-h rate (48 +/- 3% of instilled volume) that we observed in any intact species. The removal of the instilled fluid from the lung (expressed as lung liquid clearance; 0.96 +/- 0.3 ml/h) was twice as fast as the rate of alveolar and lung liquid clearance reported in the isolated or in situ rat lung models. The rate of alveolar and lung liquid clearance in these intact rats was significantly faster than those in prior studies in dogs and sheep and was similar to the rates in rabbits.

To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats. To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 Ci of 125 I-human albumin, 24 h after intratracheal instillation of bacteria. The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance. Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group. The results with each method were identical. We found that P. aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls ( P Ͻ 0.05). In both studies, this increase was inhibited with amiloride. However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance. An antitumor necrosis factor-␣ neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia ( P Ͻ 0.05). Also, TNF ␣ (5 g) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats ( P Ͻ 0.05). In normal rats instilled with TNF ␣ , propranolol had no inhibitory effect. In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNF ␣ -dependent mechanism. This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung. ( J. Clin. Invest. 1997. 99:325-335.)

Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle ␣-actin (SM-␣-actin) expression through activation of the SM-␣-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-␣-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-␣-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVPstimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely supershifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM-␣-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases.

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. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.Pulmonary hypertension (PH) and related vascular pathologies are characterized by changes in the structure of the arterial wall. These changes are largely due to the proliferation and hypertrophy of smooth muscle cells (SMCs) and increased SMC deposition of extracellular matrix in the vessel wall. The proliferation and hypertrophy of SMCs are stimulated by growth factors and proinflammatory agents such as plateletderived growth factor BB (PDGF-BB), insulin-like growth factors I and II, epidermal growth factor, basic fibroblast growth factor, vascular endothelial growth factor, endothelin-1, and thrombospondin-1, which are produced by endothelial cells, SMCs, fibroblasts, and platelets in response to vascular injury (6,11,14,15,46,59). Binding of these growth factors to their respective receptors activates associated tyrosine kinases, G proteins, and C-type phospholipases. Activation of receptor tyrosine kinases stimulates mitogen-activated protein kinase (MAPK) signaling cascades, with PDGF-BB stimulation of extracellular signal-regulated kinase 1 (ERK1)/ERK2 being a widely studied example (23, 44). G protein-coupled receptors may regulate numerous signaling pathways, with recent studies implicating RhoA/Rho kinase signaling in SMC growth and migration (52). These signaling pathways modulate the activity of downstream effectors of growth such as cyclin-dependent kinases (42) and immediate-early genes (49).These growth-promoting pathways are normally restrained in healthy arteries by endogenous mediators such as prostacyclin and NO. These agents exert antiproliferative effects on SMCs largely by increasing intracellular levels of cyclic nucleotides (53, 54), which stimulate the activity of protein kinase A (PKA) and GMP-stimulated protein kinase. Many compounds that activate adenyl cyclase (39), inhibit phosphodiesterases (50), or mimic cyclic AMP (cAMP)/cGMP (34) exert antiproliferative effects on SMC growth. Interestingly, many drugs and therapeutic agents that reduce SMC proliferation act by increasing intracellular cAMP levels (22,27,44,64). There is now substantial evidence that cAMP/PKA signaling acts as a molecular gate to block MAPK-induced proliferation in response to mitogens such as PDGF (5,23,30,44). Activation of cAMP signaling in SMCs decreases the expression of cyclin D1 and Cdk2 (60), increases the expression of antiproliferative molecules such as p53 and p21 (25), and increases overall sensitivity to antiproliferative stimuli.Given the potent proliferation-suppressing action of cAMP on SMCs, we hypothesized that the transcription factor CREB,