Proteolytic Activity and Hypoxic Pulmonary Vascular Disease

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While some studies have shown that increased activity of metalloproteinases is prevalent during the regression ofvascular disease (48) but inhibition of metalloproteinases appears to aggravate experimental pulmonary hypertension in rats (50). Our group has focused on the specific contribution of

Figure 3. A and B: Muscularization of distal pulmonary arteries in mice hypoxic for 26 days. Lung sections were immunostained for SMC a-actin before hematoxylin staining. A: Fully muscularized artery associated with alveolar duct of a nontransgenic hypoxic mouse. Arrows denote alveolar duct-associated arteries. B: Morphometric analyses of fully plus partially muscularized pulmonary arteries (15-50 |xm external diameter) associated with alveolar ducts. *P<0.004 vs normoxia. tP=0.035 vs nontransgenic hypoxia. C: Loss of distal pulmonary arteries in mice subjected to chronic hypoxia for 26 days. Lung sections were stained with van Gieson elastin stain. Mean±SE of arteries per 100 alveoli are presented. *P<0.02 vs normoxia. ■fP<0.02 vs nontransgenic hypoxia. D: Increase in RV pressure in mice subjected to acute (10% 02 for 15 min) or chronic hypoxia (26 days). Mean±SE. *P<0.01 vs normoxic, nontransgenic acute hypoxia. fP<0.004 vs nontransgenic chronic hypoxia (Modified from Ref. 56).

Figure 3. A and B: Muscularization of distal pulmonary arteries in mice hypoxic for 26 days. Lung sections were immunostained for SMC a-actin before hematoxylin staining. A: Fully muscularized artery associated with alveolar duct of a nontransgenic hypoxic mouse. Arrows denote alveolar duct-associated arteries. B: Morphometric analyses of fully plus partially muscularized pulmonary arteries (15-50 |xm external diameter) associated with alveolar ducts. *P<0.004 vs normoxia. tP=0.035 vs nontransgenic hypoxia. C: Loss of distal pulmonary arteries in mice subjected to chronic hypoxia for 26 days. Lung sections were stained with van Gieson elastin stain. Mean±SE of arteries per 100 alveoli are presented. *P<0.02 vs normoxia. ■fP<0.02 vs nontransgenic hypoxia. D: Increase in RV pressure in mice subjected to acute (10% 02 for 15 min) or chronic hypoxia (26 days). Mean±SE. *P<0.01 vs normoxic, nontransgenic acute hypoxia. fP<0.004 vs nontransgenic chronic hypoxia (Modified from Ref. 56).

serine elastase activity to the progression and regression of pulmonary vascular disease. We showed that there is heightened activity of a serine elastase 2 days after exposing rats to chronic hypobaric hypoxia and that inhibition of elastase by infusion of elastase inhibitors will greatly ameliorate the severity of pulmonary hypertension as well as the associated structural abnormalities which include extension of muscle into normally non-muscular peripheral arteries, medial hypertrophy of muscular arteries and reduction in the number of small peripheral arteries (28). Similar repression of disease is observed in a transgenic mouse in which there overexpression of the naturally occurring serine elastase inhibitor, elafin, is targeted to the vasculature under the regulation of the preproendothelin promoter (Fig. 3) (56). In unpublished studies we showed that regression of hypoxia-induced vascular disease in rats, which occurs upon return to room air, is accompanied by smooth muscle cell apoptosis. We reported that induction ofsmooth muscle cell apoptosis via elastase inhibition can reverse the fatal form of pulmonary hypertension induced by monocrotaline.

Vascular Remodeling, Elastase, MMP, and Ten a s ein

Vascular Remodeling, Elastase, MMP, and Ten a s ein

SMC Proliferation, Hypertrophy, and CT Synthesis

SMC Proliferation, Hypertrophy, and CT Synthesis

Figure 4. Schema showing how an elastase can induce changes in matrix proteins resulting in smooth muscle cell proliferation and migration.

Serum Factors Signal Elastase Transcription Serum Factor(s) - Receptor Complex

Figure 5. Schema showing how serum factors signal elastin gene transcription. Serum factors including apoAl induce MAP kinase activity, leading to nuclear translocation of AML1, and increase in elastin gene transcription.

Figure 6. Influence of NO donors and a cGMP mimetic and inhibitor on SMC elastase activity. Elastase activity was evaluated by measured solubilized [3H]-elastin in the culture medium after 24 hrs of incubation. A: Serum-starved SMCs were maintained serum free or incubated with serum-treated elastin (STE) after pretreatment with the NO donor SNAP (0.1-1 mM) for 30 min. B: Comparison of SMC elastase activity under control conditions or stimulated by serum-treated elastin (STE) after pretreatment with NO donors DETA NONOate (0.1-1 mM), SNAP (1 mM), and the cGMP mimetic 8-pCPT-cGMP (1 mM) for 30 min. The effect of pretreatment with the PKG inhibitor Rp-8-pCPT-cGMP (20 p.M) on reversing SNAP suppression of elastase activity was also evaluated. Means±SE. *P<0.05 vs. the control; fP<0.05 vs. STE with no pretreatment; tPO.OS between designated groups. C and D: Influence of NO donors, cGMP mimetic and inhibitor, and peroxynitrite (ONOO") on ERK phosphorylation. Serum-starved cells pretreated with DETA NONOate, SNAP (0.1-1 mM), 8-pCPT-cGMP, ONOO "(0.1 mM), and an inactivated negative control for ONOO" (0.1 mM) (C) or retreated with SNAP (1 mM) in the presence or absence of coadministration with Rp-8-pCPT-cGMP (20 ^M) (D) were stimulated by STE or control elastin for 5 min. Cell lysates (10 ng) were analyzed by SDS-PAGE and immunoblotted with phospho-specific or -nonspecific ERK antibodies. Means ± SE (n=3 experiments). *P< 0.05 vs. the control elastin; tP< 0.05 vs. STE with no pretreatment; JP<0.05 between designated groups (Modified from Ref. 32).

In our laboratory we are currently investigating a transgenic mouse in which the response to chronic hypoxia produces more severe pulmonary hypertension that does not regress upon return to room air. The most striking feature is the failure of new growth of peripheral arteries. The mechanisms involved are being by correlating this phenotype with the genotype as determined by a micro-array approach.

Figure 7. Hypothetical model for the regulation and function of Tenascin-C (TN-C) in vascular SMC. A: Vascular SMC attach and spread over native type-I collagen using (U integrins. Under serum free conditions, the cells withdraw from the cell cycle and become quiescent. B: Degradation of native type I collagen by matrix metalloproteinases (MMPs) leads to exposure of cryptic RGD sites that preferentially bind p3 subunit-containing integrins. In turn, occupancy and activation of p3 integrins signals the production of TN-C. C: Incorporation of multivalent TN-C protein into the underlying substrate leads to further aggregation and activation of ^-containing integrins (a|}3), and to the accumulation of tyrosine-phosphoiylated (Tyr-P) signaling molecules and actin into a focal adhesion complex. Even in the absence of the EGF ligand, the TN-C-dependent reorganization of the cytoskeleton leads to clustering of actin-associated EGF-Rs.

Figure 7. Hypothetical model for the regulation and function of Tenascin-C (TN-C) in vascular SMC. A: Vascular SMC attach and spread over native type-I collagen using (U integrins. Under serum free conditions, the cells withdraw from the cell cycle and become quiescent. B: Degradation of native type I collagen by matrix metalloproteinases (MMPs) leads to exposure of cryptic RGD sites that preferentially bind p3 subunit-containing integrins. In turn, occupancy and activation of p3 integrins signals the production of TN-C. C: Incorporation of multivalent TN-C protein into the underlying substrate leads to further aggregation and activation of ^-containing integrins (a|}3), and to the accumulation of tyrosine-phosphoiylated (Tyr-P) signaling molecules and actin into a focal adhesion complex. Even in the absence of the EGF ligand, the TN-C-dependent reorganization of the cytoskeleton leads to clustering of actin-associated EGF-Rs.

Cell culture studies in our laboratory have been carried out to explain how elastase might be regulated in pulmonary artery smooth muscle cells and how the activity ofthis enzyme may lead to pulmonary vascular disease (Fig 4).

We have shown that plasma factors that could be present in high concentration if the subendothelium when here is endothelial perturbation, can induce production of the elastase enzyme by smooth muscle cells. The mechanism requires activity of mitogen activated protein (MAP) kinases specifically extracellular regulated kinase 1 and 2 (ERK1/2) (20,21,46). This leads to increased expression and DNA binding of the transcription factor AML1 (32) which is required for the activity of elastase (Fig. 5) (53). Studies by our group showed that NO donors repress phosphorylation of ERK1/2 and the consequent expression and DNA binding of AML1 and elastase activity (Fig. 6) (32). This suggests a mechanism whereby NO may influence not only the vasoactive response to chronic hypoxia but also the structural remodeling. Increased production of elastase causes release of growth factors such as FGF-2 in a mitogenically active form resulting in smooth muscle cell proliferation (47). These growth factors can activate metalloproteinases, and augmentation of the proteolytic cascade leads to upregulation of tenascin C which in turn amplifies the proliferative response to growth factors by inducing changes in the cytoskeleton which lead to the clustering and facilitate transactivation ofgrowth factor receptors (Fig. 7).

We reason that breakdown of elastin and other extracellular matrix protein by elastase could in this way cause abnormal proliferation of medial smooth muscle cells causing medial hypertrophy and of pericytes causing muscularization of abnormally muscular peripheral arteries. Loss of small arteries may also be a function of breakdown of the extracellular matrix and basement membrane of small vessels causing endothelial cell apoptosis. Inhibition of elastase causes smooth muscle cell apoptosis leading to regression of medial hypertrophy.

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