PMC

Tenascin protein expression. (a) Representative frozen sections of carotid arteries harvested at 10 days and showing increased immunostaining in nontransgenic versus elafin-transgenic mice throughout the thickened media of the injured artery. (b) Tenascin calculated as a percent of total intima plus media area from six nontransgenic littermates and nine elafin-transgenic mice. Bars represent mean ± SEM. ^AP = 0.003 versus nontransgenic sham. ^BP = 0.005 versus elafin transgenic–injured arteries.

Mac-3 and Ly-6G expression. (a) Representative frozen sections from injured and sham-operated carotid arteries from nontransgenic and elafin-transgenic mice harvested at 10 days showing increased immunostaining for Mac-3 antigen in the injured vessel from the nontransgenic mouse. (b) Area with Mac-3 expression was quantified. Bars represent mean ± SEM from six nontransgenic and nine elafin-transgenic littermates. ^AP = 0.0003 versus nontransgenic sham. ^BP = 0.006 versus elafin transgenic–injured arteries. (c) Representative frozen sections from injured and sham-operated carotid arteries at 10 days show increased immunostaining for a neutrophil marker Ly-6G in the nontransgenic mouse. (d) Area containing the Ly-6G staining was quantified. Bars represent mean ± SEM from nine elafin-transgenic and six nontransgenic littermates. ^CP = 0.01 versus nontransgenic sham. ^DP = 0.032 versus elafin transgenic–injured arteries.

α-Actin expression. (a) Representative frozen sections of injured and sham-operated carotid arteries harvested at 10 days and showing decreased immunostaining especially in the thickest part of the vessel wall in the nontransgenic-injured artery. (b) Area immunostained for actin was calculated as a percent of total intima plus media area. Bars represent mean ± SEM from six nontransgenic littermates and nine elafin-transgenic mice. ^AP = 0.0002 versus nontransgenic sham. ^BP = 0.0015 versus elafin transgenic–injured arteries.

Medial thickening. (a) Representative sections of carotid arteries stained with Movat’s pentachrome showing medial thickening in the nontransgenic injured compared with the sham or elafin transgenic–injured and sham vessels. (b) Graphs depict mean ± SEM at day 10 after carotid arterial injury in elafin-transgenic (n = 9) and nontransgenic (n = 6) littermates. Medial thickening is quantified by the area between the inner- and outermost elastic laminae. ^AP = 0.0004 versus nontransgenic sham arteries. ^BP = 0.036 versus elafin transgenic–injured arteries. (c) Cell numbers calculated by counting nuclei in the intima + media. ^CP = 0.0001 versus nontransgenic sham. ^DP = 0.0001 versus elafin transgenic–injured arteries. (d) Neointima formation observed in the Movat’s pentachrome stained section of an injured artery of one of the nontransgenic mice.

Elafin-overexpressing transgenic mice. (a) Diagram of the construct used to create the transgenic mice in which COOH-terminal FLAG-tagged human elafin was cloned downstream of the mouse preproendothelin promoter. Signal peptide (SP), transglutaminase substrate (TG), and elafin inhibitory domains are denoted along the top of elafin cDNA. ET-1, endothelin-1. (b) Carotid arteries from elafin-transgenic and nontransgenic mice were immunostained with anti-FLAG antibodies before hematoxylin counterstaining. Brown immunoperoxidase staining for FLAG-elafin is observed in the carotid artery from the elafin-transgenic but not the nontransgenic littermate. (c) Six hours after carotid arterial injury, sham-operated and injured arteries pooled from four different elafin-transgenic or nontransgenic mice were assayed for elastase activity. Elastase activity is expressed per artery as described in the legend to Figure . Recombinant human elafin (rElafin; 2 ng) completely abolished the elastase activity of nontransgenic-injured arteries.

Carotid arterial wire injury. (a) Representative H&E-stained sections of noninjured and injured carotid arteries (harvested 6 hours after endothelial denudation) from one of two different CD1 mice. Endothelial nuclei apparent in the noninjured vessel (arrowheads) are not seen after injury. (b) Elastase activity from pooled homogenates of three or four vessels was assayed and expressed per artery after a calculation based on a standard plot generated by using known concentrations of human leukocyte elastase. Bar at 6 hours represents an average of two separate experiments with similar values of 0.315 and 0.346 mU per artery. Recombinant human elafin (rElafin; 2 ng) completely abolished the elastase activity observed at 6 hours and 7 days after injury.