Chronic administration of minoxidil protects elastic fibers and stimulates their neosynthesis with improvement of the aorta mechanics in mice.

Arterial wall elastic fibers, made of 90% elastin, are arranged into elastic lamellae which are responsible for the resilience and elastic properties of the large arteries (aorta and its proximal branches). Elastin is synthesized only in early life and adolescence mainly by the vascular smooth muscles cells (VSMC) through the cross-linking of its soluble precursor, tropoelastin. In normal aging, the elastic fibers become fragmented and the mechanical load is transferred to collagen fibers, which are 100-1000 times stiffer than elastic fibers. Minoxidil, an ATP-dependent K+ channel opener, has been shown to stimulate elastin expression in vitro, and in vivo in the aorta of male aged mice and young adult hypertensive rats. Here, we have studied the effect of a 3-month chronic oral treatment with minoxidil (120 mg/L in drinking water) on the abdominal aorta structure and function in adult (6-month-old) and aged (24-month-old) male and female mice. Our results show that minoxidil treatment preserves elastic lamellae integrity at both ages, which is accompanied by the formation of newly synthesized elastic fibers in aged mice. This leads to a generally decreased pulse pressure and a significant improvement of the arterial biomechanical properties in female mice, which present an increased distensibility and a decreased rigidity of the aorta. Our studies show that minoxidil treatment reversed some of the major adverse effects of arterial aging in mice and could be an interesting anti-arterial aging agent, also potentially usable for female-targeted therapies.

Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice.

Surfactant protein B (SP-B) is expressed tissue specifically in the lung and is developmentally regulated. To identify genomic regions that control SP-B expression, we analyzed SP-B promoter activity in transgenic mice containing rabbit SP-B 5'-flanking DNA fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Results showed that whereas the -2,176/+39-bp fragment failed to express CAT, shorter fragments of -730/+39 and -236/+39 bp expressed CAT tissue specifically in the lung. Further deletion of 5'-flanking DNA to -136 bp resulted in no expression of CAT. Immunostaining demonstrated that both -730/+39- and -236/+39-bp regions expressed CAT specifically in alveolar type II and Clara cells. The -236/+39-bp region expressed CAT at a significantly lower level than the -730/+39-bp region. CAT expression in mice containing the -730/+39-bp region was detected in embryonic day 14 lung and attained maximum levels in day 18 lung, indicating that the developmental expression of CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within -236/+39 bp of the SP-B gene. Additionally, these data suggest that the -2,176/-730- and -730/-236-bp regions contain the DNA elements that repress and enhance SP-B gene transcription, respectively.

Functional importance of connective tissue repair during the development of experimental abdominal aortic aneurysms.

BACKGROUND: Abdominal aortic aneurysms (AAAs) involve an unfavorable balance between the destruction and the repair of connective tissue proteins. The purpose of this study was to assess the functional importance of connective tissue repair during experimental aneurysmal degeneration. METHODS: Male Wistar rats (n = 70) underwent transient intraluminal perfusion of the abdominal aorta with porcine pancreatic elastase. In Study I, the aortic diameter was measured before elastase perfusion and at days 0, 2, 7, and 14 (n = 6 rats at each interval). Aortic wall concentrations of desmosine (Des) and hydroxyproline (OHP) were measured at each interval, and the expression of tropoelastin (TE), alpha1(I) procollagen (PC), and lysyl oxidase genes was evaluated by reverse transcription-polymerase chain reaction. In Study II, 22 rats were treated with beta-aminopropionitrile (BAPN) to block connective tissue repair. In Study III (n = 30), rats were treated with doxycycline, a matrix metalloproteinase inhibitor, beginning 7 days after elastase perfusion. RESULTS: AAAs consistently developed between 7 and 14 days after elastase perfusion. Aortic wall Des concentration decreased markedly during aneurysm development, reaching 3% of normal by day 14 (377 +/- 22 pmol of Des/sample on day 0 vs 9 +/- 1 pmol of Des/sample on day 14; P <.05). Aortic wall OHP decreased to only 68% of normal at the same interval (121 +/- 10 nmol of OHP/sample on day 0 vs 82 +/- 14 nmol of OHP/sample on day 14; P <.05). TE and PC expression was undetectable in healthy aorta, but they both increased by day 7 (P <.05); while TE expression decreased again by day 14, PC continued to rise. Lysyl oxidase expression progressively decreased at all intervals after elastase perfusion. Treatment with beta-aminoproprionitrile resulted in acute aortic dissection in 81% of the rats (50% mortality). These early deaths occurred between days 3 and 6, coinciding with aortic infiltration by proteinase-secreting inflammatory cells. Delayed treatment with doxycycline suppressed the progression of aneurysmal dilatation between days 7 and 21 (P <.05 vs untreated controls). CONCLUSIONS: The development of elastase-induced AAAs is accompanied by an active process of connective tissue repair. While this reparative process is necessary to stabilize the developing aneurysm wall, it is insufficient to prevent aneurysm progression. In contrast, reducing the proteolytic destruction of connective tissue proteins promotes stabilization of the aneurysmal aorta.

Suppression of experimental abdominal aortic aneurysms by systemic treatment with a hydroxamate-based matrix metalloproteinase inhibitor (RS 132908).

BACKGROUND: Abdominal aortic aneurysms (AAAs) are associated with chronic inflammation, disruption of medial elastin, and increased local production of elastolytic matrix metalloproteinases (MMPs). The purpose of this study was to investigate how treatment with a hydroxamate-based MMP antagonist (RS 132908) might affect the development of experimental AAAs. METHODS: Male Wistar rats underwent intraluminal perfusion of the abdominal aorta with 50 units of porcine pancreatic elastase followed by treatment for 14 days with RS 132908 (100 mg/kg/day subcutaneously; n = 8) or with vehicle alone (n = 6). The external aortic diameter (AD) was measured in millimeters before elastase perfusion and at death, with AAA defined as an increase in AD (DeltaAD) of at least 100%. Aortic wall elastin and collagen concentrations were measured with assays for desmosine and hydroxyproline, and fixed aortic tissues were examined by light microscopy. RESULTS: AAAs developed in all vehicle-treated rats, with a mean AD (+/- SE) that increased from 1.60 +/- 0.03 mm before perfusion to 5.98 +/- 1.02 mm on day 14 (DeltaAD = 276.4 +/- 67.7%). AAAs developed in only five of eight animals (62.5%) after MMP inhibition, with a mean AD that increased from 1.56 +/- 0.05 mm to 3.59 +/- 0.34 mm (DeltaAD = 128.1 +/- 18.7%; P <.05, vs vehicle). The overall inhibition of aortic dilatation attributable to RS 132908 was 53.6 +/- 6.8%. Aortic wall desmosine fell by 85.4% in the vehicle-treated rats (1210.6 +/- 87.8 pmol/sample to 176.7 +/- 33.4 pmol/sample; P <.05) but only by 65.6% in the animals treated with RS 312908 (416.2 +/- 120.5 pmol/sample). In contrast, hydroxyproline was not significantly affected by either elastase perfusion or drug treatment. Microscopic examination revealed the preservation of pericellular elastin and a greater degree of fibrocollagenous wall thickening after MMP inhibition, with no detectable difference in the extent of inflammation. CONCLUSIONS: Systemic MMP inhibition suppresses aneurysmal dilatation in the elastase-induced rodent model of AAA. Consistent with its direct inhibitory effect on various MMPs, RS 132908 promotes the preservation of aortic elastin and appears to enhance a profibrotic response within the aortic wall. Hydroxamate-based MMP antagonists may therefore be useful in the development of pharmacologic approaches to the suppression of AAAs.

Chronic lung injury in preterm lambs. Disordered respiratory tract development.

The cause of chronic lung disease of early infancy, often called bronchopulmonary dysplasia (BPD), remains unclear, partly because large-animal models that reliably reproduce BPD have not been available. We developed a model of BPD in lambs that are delivered prematurely and ventilated for 3 to 4 wk after birth to determine whether the histopathology of chronic lung injury in premature lambs mimics that which occurs in preterm infants who die with BPD, and to compare two ventilation strategies to test the hypothesis that differences in tidal volume (VT) influence histopathologic outcome. The two ventilation strategies were slow, deep ventilation (20 breaths/min, 15 +/- 2 ml/kg body weight VT; n = 5) or rapid, shallow ventilation (60 breaths/min, 6 +/- 1 ml/kg body weight VT; n = 5). Lambs were delivered at 125 +/- 4 d gestation (term = 147 d), treated with surfactant, and mechanically ventilated with sufficient supplemental oxygen to maintain normal arterial oxygenation (60 to 90 mm Hg). Quantitative histologic analysis revealed lung structural abnormalities for both groups of experimental lambs compared with lungs of control term lambs that were < 1 d old (matched for developmental age; n = 5) or 3 to 4 wk old (matched for postnatal age; n = 5). Compared with control lambs, chronically ventilated preterm lambs had pulmonary histopathology characterized by nonuniform inflation patterns, impaired alveolar formation, abnormal abundance of elastin, increased muscularization of terminal bronchioles, and inflammation and edema. Slow, deep ventilation was associated with less atelectasis, less alveolar formation, and more elastin when compared with rapid, shallow ventilation. We conclude that prolonged mechanical ventilation of preterm lambs disrupts lung development and produces pulmonary histopathologic changes that are very similar to those that are seen in the lungs of preterm infants who die with BPD. This chronic lung disease is not prevented by surfactant replacement at birth, does not appear to require arterial hyperoxia, and is influenced by VT.

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

Chronic lung injury in preterm lambs: disordered pulmonary elastin deposition.

Prolonged mechanical ventilation of premature neonates is often associated with abnormal morphological development of the lung and chronic lung disease, sometimes called bronchopulmonary dysplasia (BPD). Impaired alveolar development is a hallmark of this disease. To better understand the effects of mechanical ventilation on lung elastin expression, we studied lung tissue from 10 preterm lambs (gestation = 125 days; term = 148 days) mechanically ventilated for 3-4 wk at a respirator rate of 20 breaths/min and tidal volume of 15 +/- 5 ml/kg (n = 5) or 60 breaths/min and tidal volume of 5 +/- 2 ml/kg (n = 5). Histopathology showed increased elastin accumulation and abnormal morphological development in the ventilated groups. Postmortem lung desmosine content was increased significantly in the 20 breaths/min group. Tropoelastin mRNA expression was increased in both ventilated groups. In situ hybridization localized increased tropoelastin mRNA expression to sites of accumulated elastin in extended alveolar walls with scant, attenuated secondary crests. Lung collagen content, as assessed by the amount of hydroxyproline in lung tissue, was similar to controls. These data suggest that excessive production and accumulation of elastin is associated with chronic lung injury from prolonged mechanical ventilation after premature birth.

Morphological and biochemical studies of the elastic fibres in the craniomandibular joint articular disc of the tight-skin mouse.

The tight-skin (TSK) mouse is characterized by the hyperplasia of loose connective tissues, and of excessive growth of cartilage and of bones including the mandible. Since the fibroelastic connective tissues of the craniomandibular joint (CMJ) are essential to the functions of this joint, the present histological study compared the presence and general distribution of elastic fibres in CMJ discal tissues of TSK and normal mice. The excised CMJs were processed for light microscopy. The tissues were fixed, demineralized, embedded in paraffin, sectioned and then stained with resorcin-fuchsin to demonstrate elastic fibres. There were no obvious histological differences in either the amount or the distribution of elastic fibres in the discs from the two groups. In both groups, elastic fibres were found in the disc and in many of the attachments of the disc to the mandible and squamosal bone. In addition to the morphological preparations, articular discs and samples of lung tissue were excised from other mice and subjected to a radioimmunoassay for desmosine in order to estimate the amounts of elastin in these tissues; the amount of elastin was significantly reduced in the TSK lung, but the amounts of elastin in the TSK and normal CMJ discal tissues were not significantly different statistically. These morphological and histochemical results suggest that the distribution and quantity of elastic fibres in the TSK mouse disc are not significantly different from those in the normal mouse articular disc. Moreover, these data may be interpreted to either suggest a differential effect on the elastic fibres in different TSK tissues, or to support the suggestion that abnormal degradation of elastic fibres may not be characteristic of the TSK mouse.

Secretory leukoprotease inhibitor attenuates lung injury induced by continuous air embolization into sheep.

Continuous air embolization (CAE) into the pulmonary arterial circulation of sheep results in functional and structural changes of chronic pulmonary hypertension. Release of elastin peptides into lung lymph during CAE and attenuation of CAE-induced pulmonary hypertension by neutrophil depletion suggest that neutrophil elastase may contribute to these changes. To investigate this notion, we treated awake sheep with a potent neutrophil elastase inhibitor, recombinant secretory leukoprotease inhibitor (SLPI) (100 mg/day by aerosol), during 12 days of CAE (CAE+SLPI; n = 7). Controls included sheep receiving CAE + vehicle (VEH) (n = 6), VEH alone (n = 3), and SLPI alone (n = 3). SLPI significantly attenuated the CAE-induced increases in lung lymph flow (day 8; 2.3 +/- 0.5 vs. 5.6 +/- 1.7 ml/15 min), protein clearance (day 8; 1.36 +/- 0.32 vs. 3.08 +/- 0.84 ml/15 min), and elastin peptide concentration (day 8; 243 +/- 41 vs. 398 +/- 44 ng/ml). SLPI delayed the onset of sustained pulmonary hypertension from day 8 to day 12. Both CAE groups showed similar structural changes in the pulmonary arteries. SLPI was well tolerated in control sheep and did not affect hemodynamics or structure. We conclude that serine proteases may contribute to the early initiation of chronic pulmonary hypertension but do not play a striking role in its eventual development.

Composition and mechanics of mesenteric resistance arteries from pregnant rats.

We tested the hypothesis that the systemic resistance vasculature of the rat is remodeled during pregnancy as evidenced by significant alterations in the passive mechanical properties and extracellular matrix proteins in mesenteric arteries. Mechanical characteristics were determined for arteries from 20-day pregnant rats (n = 6) and age-matched controls (n = 5). Lumen diameter and wall thickness were measured in pressurized arteries (250-microns diameter) using a dimension analyzing system. Distensibility (the relative change in diameter per unit change in pressure) was less in the arteries from the pregnant rats (P less than 0.01). The calculated stress-strain relationships and elastic moduli indicated that the arteries were less stiff by late gestation (P less than 0.05). Ultramicro amino acid analysis and radioimmunoassay were used to measure hydroxyproline, desmosine, and leucine as indicators of collagen, elastin, and total protein, respectively, in similar-sized arteries. Hydroxyproline/leucine (index of collagen) and desmosine/leucine (elastin concentration) decreased 19 and 15% by late gestation (P less than 0.05). The significant alterations in passive mechanics and in extracellular protein content support the concept that arterial wall remodeling in the peripheral vasculature may be one component of the cardiovascular adaptations during pregnancy.

Risk factors for the degradation of lung elastic fibers in the ventilated neonate. Implications for impaired lung development in bronchopulmonary dysplasia.

In order to evaluate the risk for proteolytic destruction of lung parenchymal elastic fibers in ventilated premature infants, the concentrations of elastase were determined in tracheal aspirates of 65 infants from whom we obtained a total of 327 sequential samples. Elastase was detected at least once in 39 of the 65 infants studied. Eleven of these infants were ventilated with greater than 60% oxygen for greater than 5 days. In 19 infants, the presence of elastase was associated with positive bacterial and/or viral cultures and/or elevated ratios (greater than 0.22) of immature neutrophils to total neutrophils. Elastase was not detected in the lung secretions of 26 infants ventilated with greater than 60% oxygen for less than 3 days, suggesting minimal risk for elastic fiber destruction in the intubated infant who neither has pneumonia nor requires prolonged hyperoxic ventilation. The risk for elastic fiber destruction was further evaluated by analyzing sequential urine and tracheal aspirate samples for the presence of an elastolytic degradation product of elastin (desmosine). The biochemical data indicated a potential risk for proteolytic destruction of elastic fibers in association with infection and/or prolonged hyperoxic exposure. In addition, autopsy specimens obtained from three of the infants revealed structurally abnormal lung parenchymal elastic fibers. Because elastic fibers are believed to provide the structural support for alveolar septal development, proteolytic degradation of these fibers may be a significant factor in the impaired lung development that occurs in infants with bronchopulmonary dysplasia.

In vitro effects of elastase on rabbit craniomandibular joint articular disks.

This in vitro study correlates morphologic and radioimmunoassay (RIA) findings on the effects of elastase on the elastic fibers that are found in the rabbit craniomandibular joint (CMJ) articular disk. Articular disks were removed from rabbit CMJs at necropsy, and cut sagittally into two pieces which were incubated in 0.3 ml of phosphate-buffered saline containing either 0, 12.5, 25 or 50 units of porcine pancreatic elastase for either 1, 3 or 24 h. The quantitative RIA findings correlated well with the qualitative light-microscopic observations in that both methods showed a reduction in the amounts of elastin in the CMJ disks following enzyme treatment. However, the morphologic appearance of most of the elastase-treated disks suggested that the destruction of the elastic fibers was more extensive than was suggested by the results of the RIA which indicated that some elastin remained in the tissues of the disks even when the highest enzyme level and longest incubation period were combined. The results of this study also support the interpretation that the resorcin-fuchsin-stained fibers in the rabbit CMJ disk are elastic fibers.

Effect of ultrasound therapy on the repair of Achilles tendon injuries in rats.

The purpose was to determine the effects of selected regimens of ultrasound therapy on the rates of repair of injured Achilles tendons of rats. Specific dependent variables examined were tendon breaking strength and rate of collagen formation. A puncture technique was used to induce injuries to both Achilles tendons of rats. Continuous ultrasound was administered to the left tendon for 4 min per treatment session at an intensity of 1.5 W.cm-2. Rats were sacrificed 2, 5, 9, 15, and 21 d following injury for measurement of tendon breaking strength and 3 and 5 d postinjury for analysis of collagen synthesis. Breaking strength was defined as the minimum force required to completely rupture the tendon. Collagen synthesis was indicated by the conversion of labeled proline to hydroxyproline. The breaking strengths of the treated tendons were significantly greater than strengths of the untreated tendons 5, 9, 15, and 21 d postinjury. Collagen synthesis was increased in the treated tendons compared with the untreated tendons 5 d postinjury. The results indicate that ultrasound treatment increases the rate of repair of injured Achilles tendons of rats. The results are also consistent with an association between increased collagen synthesis and greater breaking strength during tendon repair.

Morphological and biochemical evidence for elastic fibres in the Syrian hamster temporomandibular joint disc.

Elastic fibres are considered to be important for the normal biomechanical functions of the TMJ. The objective here was to correlate morphological evidence for the presence of elastic fibres in discal tissues with biochemical evidence for elastin. For light microscopy, the joints were removed en bloc, processed for paraffin embedding, sectioned and stained with resorcin-fuchsin. For biochemical study, a radioimmunoassay for desmosine was used to estimate the amount of elastin in excised articular discs. The histological preparations showed that numerous elastic fibres were present in various areas of the disc and in some of the discal attachments to surrounding bone. Radioimmunoassay also indicated that elastin was present in these tissues. Therefore, the biochemical findings support the morphological in suggesting that elastic fibres are present in the articular disc of the hamster TMJ.

Bronchoalveolar lavage desmosine in bleomycin-induced lung injury in marmosets and patients with adult respiratory distress syndrome.

Measurement of urinary desmosine in experimental models of emphysema has been used to demonstrate elastin catabolism. In order to evaluate the hypothesis that accelerated elastin degradation also occurs in association with acute lung injury characterized by fibrotic repair, we prepared acid hydrolysates of lung lavage (LL) and used a radioimmunoassay for desmosine to measure concentrations of this elastic-specific cross-link in LL. Lavage desmosine (pmol/100 microliter LL) was measured following bleomycin-induced lung injury in marmosets and was shown to be elevated at 1 week (median 6.0, range 5.1-7.8), 2 weeks (8.4, 6.2-8.7), and 4 weeks (7.6, 4.8-7.8) compared to control levels (1.8, 1.4-3.7). Elevations of lavage desmosine after bleomycin were temporarily associated with remodeling of the lung as indicated by increased total lung collagen, reduced diffusing capacity and lung compliance, and histologic evidence of pulmonary fibrosis. Bronchoalveolar lavage (BAL) desmosine was measured in patients with the Adult Respiratory Distress Syndrome (ARDS) and compared with patients at risk, patients with other interstitial lung diseases, and normal healthy controls. BAL desmosine (pmol/100 microliters) was not significantly different in patients with ARDS (3.2, 2.1-3.0), patients at risk for ARDS (2.8, 2.5-4.4), and those with interstitial lung disease (3.0, 1.7-5.3) compared to normal controls (2.9, 1.9-4.7). There were poor correlations of BAL desmosine with physiologic indices of severity of disease in patients with ARDS and those at risk. Accelerated elastolysis occurred in the lower respiratory tract during the evaluation of bleomycin-induced pulmonary fibrosis in marmosets but was undetectable in BAL of patients studied within the first 3 days of ARDS.

Degradation of elastin in experimental elastase-induced emphysema measured by a radioimmunoassay for desmosine.

Experimental emphysema, produced by a single intratracheal injection of elastase in hamsters, progresses in severity over months. To investigate whether this progression is due to continuous elastolysis, we measured the urinary excretion of desmosine by radioimmunoassay (RIA) as a measure of elastin catabolism in vivo. Normal hamster excreted 1.6 microgram of desmosine, equivalent to a daily turnover of approximately 0.4 mg of elastin. During the first 24 hr after injection of 25 units of elastase, excretion of desmosine was increased threefold, rapidly returning to normal over several days. Desmosine excretion was normal after 6 days. Homogenates of lungs from elastase-injected hamsters were incubated in vitro, and the release of soluble desmosine was followed by RIA as a measure of the active elastase in the tissue. The method was sufficiently sensitive to detect 0.1 microgram of enzyme bound to elastin. Desmosine solubilized in vitro from lungs removed at intervals after elastase injection was 10-fold that of control at 1 hr and slightly elevated at 48 hr, but equaled control levels at 7 days. These results indicate that the late progression of elastase-induced emphysema is not accompanied by increased elastolysis.