Elastin in a neonatal rat smooth muscle cell culture has greatly decreased susceptibility to proteolysis by human neutrophil elastase. An in vitro model of elastolytic injury.
A neonatal rat aorta smooth muscle cell culture system with a unique elastin-rich extracellular matrix was used as a model substrate for elastases. To study the susceptibility to solubilization of insoluble elastin, cultures were incubated in the presence of human neutrophil elastase (HNE) or porcine pancreatic elastase (PPE) and in the absence of serum for periods up to 45 min. Both the incubation media and cell layers were then assessed for elastin and collagen markers, total protein, and lactate dehydrogenase (LDH). Although HNE and PPE exhibited comparable activity against elastin purified from the cell layer, HNE exhibited a 6.7- to 25-fold reduction in its elastin solubilizing activity using intact cell layers as compared with the purified elastin, whereas PPE exhibited only a 1.5- to 2.5-fold reduction. This effect could not satisfactorily be explained as preferential inhibition of HNE activity in the culture system, because the amount of protein solubilized by HNE was 59% that of PPE. The mean elastin content of PPE-solubilized protein was 110% that of the elastin content of the corresponding cell layer; the value for HNE-solubilized protein was only 16%. Thus, the amount of elastin per microgram of solubilized protein for HNE was 15% that for PPE. Possible explanations for the greatly diminished elastolytic activity of HNE in the culture system include the preference of HNE for other substrates in the cell layer, the inability of HNE to penetrate sufficiently into the cell layer, and the presence of sulfated glycosaminoglycans in the vicinity of the elastin that act in an inhibitory fashion. Although there was extensive proteolytic damage to the extracellular matrix, LDH and DNA measurements indicated that little loss of cells or cell viability occurred. The observed differences in elastolytic activity of HNE and PPE in the culture system parallel the relative emphysema-inducing potency of the elastases in the hamster model of elastase-induced emphysema.
Stone
P J
PJ
Boston University School of Medicine, Department of Biochemistry, Massachusetts 02118.
McMahon
M P
MP
Morris
S M
SM
Calore
J D
JD
Franzblau
C
C
D000818:Animals; D000831:Animals, Newborn; D001011:Aorta / Q000166:cytology; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D004549:Elastin / Q000302:isolation & purification / Q000378:metabolism*; D005109:Extracellular Matrix / Q000032:analysis; D006801:Humans; D009130:Muscle, Smooth / Q000032:analysis / Q000166:cytology*; D009504:Neutrophils / Q000201:enzymology*; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013045:Species Specificity; D013379:Substrate Specificity; D013552:Swine
D003094:Collagen; D004549:Elastin; D010196:Pancreatic Elastase