Reduction in vascular lesion formation by hirudin secreted from retrovirus-transduced confluent endothelial cells on vascular grafts in baboons.

BACKGROUND: The hypothesis that thrombin mediates the formation of neointimal vascular lesions at sites of mechanical vascular injury has been tested in baboons by measurement of the effects of hirudin delivered by retrovirus-transduced hirudin-secreting vascular endothelial cells (ECs) lining surgically implanted arterial vascular grafts (AVGs). METHODS AND RESULTS: The antithrombotic efficacy of baboon ECs transduced with cDNA encoding hirudin was assessed in vitro and in vivo on thrombogenic segments in chronically exteriorized femoral arteriovenous (AV) shunts. Bilateral brachial AVGs lined with hirudin-transduced versus nonhirudin control ECs at confluent density were surgically implanted, and vascular lesion formations at distal graft-vessel anastomoses were compared after 30 days. Hirudin-transduced ECs secreted 20+/-6 ng x 10(6) cells(-1) x 24 h(-1) (range, 14 to 24 ng x 10(6) cells(-1) x 24 h(-1)) hirudin in supernatants of static cultures. Hirudin-secreting ECs on segments of collagen-coated graft interposed in chronic AV shunts decreased the accumulation of (111)In-labeled platelets to 0.52+/-0.34 x 10(9) platelets, compared with 0.82+/-0.49 x 10(9) platelets in controls (P = 0.03) and reduced platelet deposition in propagated thrombotic tails extending downstream from segments of vascular graft from 1.38+/-0.41 x 10(9) platelets in controls to 0.59+/-0.22 x 10(9) platelets (P = 0.04). ECs recovered from 30-day AVG implants generated 17+/-9 ng x 10(6) cells(-1) x 24 h(-1) (range, 9 to 25 ng x 10(6) cells(-1) x 24 h(-1)) hirudin. Hirudin-secreting ECs reduced neointimal lesion formation at distal graft-vessel anastomoses, ie, 1.02 mm(2) (range, 0.88 to 1.95 mm(2)) versus 1.82 mm(2) (range, 0.88 to 2.56 mm(2)) in contralateral AVGs bearing nonhirudin control ECs (P<0.01). CONCLUSIONS: Viral vector-directed secretion of hirudin from ECs lining implanted AVGs significantly reduces the formation of thrombus and neointimal vascular lesions.

Fibrocellular tissue responses to endovascular and external beam irradiation in the porcine model of restenosis.

PURPOSE: Endovascular radiation has reduced postangioplasty restenosis in preclinical and early clinical studies. External radiation treatment may have advantages over endovascular therapy. We examined vascular and perivascular tissue responses to endovascular and external irradiation in pig coronary arteries. METHODS AND MATERIALS: Ninety-one animals received endovascular or external radiation following balloon injury and were sacrificed at 14, 30, or 180 days. Injured segments of coronary vessels including perivascular and myocardial tissues were evaluated with histochemistry. RESULTS: Endovascular radiation was associated with delayed arterial wound healing as late as 6 months, evidenced by paucity of smooth muscle alpha-actin in neointimal cells compared to control. External treatment was associated with increased collagen in neointima and adventitia, and focal interstitial necrosis in adjacent myocardium. CONCLUSIONS: These investigations showed whole-heart 14 Gy external radiation treatment following coronary injury exacerbated certain aspects of arterial healing. In addition focal myocardial necrosis and fibrosis was observed following external but not endovascular irradiation. Endovascular radiation has some advantages over external irradiation; however the persistence of a synthetic smooth muscle cell phenotype in the neointima at 6 months suggests ionizing radiation in general may have profound effects on vessel architecture over the long term.

The role of alpha and beta platelet-derived growth factor receptor in the vascular response to injury in nonhuman primates.

Restenosis remains a significant clinical problem associated with mechanical interventional procedures for arterial revascularization or repair, including coronary angioplasty and stenting. Studies with rodents have established that platelet-derived growth factor (PDGF), a potent chemotactic and mitogenic agent for vascular smooth muscle cells, is a key mediator of lesion formation after vascular injury. To further explore this hypothesis in a more clinically relevant model, neutralizing monoclonal antibodies (mAbs) were used to examine the effect of selective inhibition of alpha or beta PDGF receptor (PDGFR) on neointima formation in nonhuman primates. Carotid arteries were injured by surgical endarterectomy and femoral arteries by balloon catheter dilatation. Immunostaining revealed that both injuries induced cell proliferation and the upregulation of beta PDGFR but not alpha PDGFR. By 7 days after injury, beta PDGFR staining was limited to the luminal region of the media, the small areas of neointima, and the adventitia. Nearly all bromodeoxyuridine-positive cells were found in these regions as well. After 30 days, a concentric neointima that stained strongly for beta PDGFR had formed in the carotid and femoral arteries. Treatment of baboons with anti-beta PDGFR mAb 2A1E2 for 6 days after injury reduced the carotid artery and femoral artery lesion sizes by 37% (P<0.05) and 48% (P<0.005), respectively, when measured at 30 days. Under the same conditions, treatment with anti-alpha PDGFR mAb 2H7C5 had no effect. These findings suggest that PDGF mediates neointima formation through the beta PDGFR, and that antagonism of this pathway may be a promising therapeutic strategy for reducing clinical restenosis.

Remodeling after myocardial infarction in humans is not associated with interstitial fibrosis of noninfarcted myocardium.

OBJECTIVES: This study was specifically designed to evaluate whether noninfarcted hypertrophic myocardium in patients with end-stage heart failure after myocardial infarction (MI) is associated with an increase in interstitial fibrous tissue. BACKGROUND: Postinfarction remodeling consists of complex alterations that involve both infarcted and noninfarcted myocardium. The question arises whether ventricular dysfunction is due to physical events, such as inadequate myocardial hypertrophy to compensate for increased tangential wall stress, or is caused by the development of progressive interstitial fibrosis in noninfarcted myocardium. METHODS: Fifteen hearts were obtained as cardiac explants (n = 13) or at autopsy (n = 2) from patients with end-stage coronary artery disease. Sixteen normal hearts served as reference hearts. Samples were taken from the left ventricular (LV) wall that contained the infarcted area, the border area and noninfarcted myocardium remote from scar areas. Collagen was quantified biochemically and microdensitophotometrically. Collagen type I and III ratios were analyzed by using the cyanogen bromide method and immunohistochemical staining, followed by microdensitophotometric quantification. RESULTS: In noninfarcted myocardium remote from the scar areas, total collagen levels and collagen type I/III ratios did not differ statistically from those in reference hearts. These observations contrasted with high total collagen content and high collagen type I/III ratios in scar and border areas. CONCLUSIONS: Remodeling of LV myocardium after MI in patients with end-stage heart failure is not necessarily associated with interstitial fibrosis in noninfarcted hypertrophic myocardium remote from scar areas. This finding raises questions regarding therapeutic interventions designed to prevent or retard the development of interstitial fibrosis.

Multiple labeling in electron microscopy: its application in cardiovascular research.

The heart is a muscular pump kept together by a network of extracellular matrix components. An increase in collagens, as in chronic congestive heart failure (CHF), is thought to have a negative effect on cardiac compliance and, thus, on the clinical condition. Conventional electron microscopy allows for the study of cellular and extracellular components and scanning electron microscopy (SEM) can put these structures in three-dimensional perspective. However, in order to study extracellular matrix components in relation to cells, immunoelectron microscopy is superior. We have used this technique in our studies on heart failure. Heart specimens were fixed in 4% paraformaldehyde and 0.1% glutaraldehyde in sodium cacodylate buffer, dehydrated by the method of progressive lowering of temperature and embedded in LR Gold plastic. Immunolabeling could be achieved with different sized gold-conjugated secondary antibodies or protein-A gold conjugates. Depending on the objective, ultra small gold (USG) conjugates or a regular probe size can be used. Labeling efficiency could be increased by bridging antibodies. The double and triple staining procedures were based on single staining methods using one- and two-face labeling. The choice of antibodies and gold conjugates depended on the objectives. Immunoelectron microscopy, using multiple labeling, allowed a detailed study of the organization of the extracellular matrix and its relationship with cardiac myocytes. This may prove to be a useful tool for the study of chronic heart failure.

Chronic congestive heart failure is associated with a phenotypic shift of intramyocardial endothelial cells.

BACKGROUND: There is evidence that patients with chronic congestive heart failure have endothelial cell-related abnormalities of the peripheral circulation and the coronary microvasculature. For that reason, we have studied the phenotypic expression of endothelial cells in hearts of patients with congestive heart failure. METHODS AND RESULTS: We studied cardiac explants (n = 19) and autopsy hearts (n = 5) of patients with chronic congestive heart failure caused by either a dilated cardiomyopathy (n = 12) or ischemic heart disease (n = 12) and compared them with normal hearts (n = 12). The antigenic expression obtained with several endothelial cell markers (factor VIII-related antigen, EN-4, Ulex europaeus agglutinin-1 (UEA-1), PAL-E, endoglin, and endothelin) and adhesion molecules (intercellular adhesion molecule [ICAM], vascular cell adhesion molecule [VCAM], or E-selectin) was compared by use of immunohistochemical techniques. On the basis of the initial findings, the number of PAL-E- and EN-4-positive vessels was counted. The incidence of PAL-E-positive vessels per area was quantified and related to the percentage of heart muscle cells and the total number of vessels per area. In control hearts, endothelial cells rarely were positive for PAL-E. In hearts of patients with ischemic cardiomyopathies, there was distinct staining with this marker. Hearts of patients with dilated cardiomyopathies showed a marked increase in the number of PAL-E-positive endothelial cells. Vessels with a muscular media were PAL-E-negative. Two-sample analysis revealed a statistically significant difference between hearts with dilated cardiomyopathies and ischemic cardiomyopathies (P < .01), between hearts with dilated cardiomyopathies and control hearts (P < .01), and between hearts with ischemic cardiomyopathies and control hearts (P < .01). Endoglin and ICAM were positive but nondiscriminating. Endothelin, VCAM, and E-selectin were negative. CONCLUSIONS: A phenotypic shift in endothelial antigen expression of the coronary microvasculature occurs in both ischemic hearts and hearts with dilated cardiomyopathies, as revealed by PAL-E, compared with control hearts. The change may relate to compensatory mechanisms in long-standing chronic heart failure.

Dilated cardiomyopathy is associated with an increase in the type I/type III collagen ratio: a quantitative assessment.

OBJECTIVES: The aim of this study was to quantify total collagen and the type I/type III collagen ratio and their localization in hearts with dilated cardiomyopathy. BACKGROUND: Patients with dilated cardiomyopathy have an increase in intramyocardial fibrillar collagen. Types I and III are the main constituents and have different physical properties that may affect cardiac compliance. METHODS: Nineteen hearts with dilated cardiomyopathy were studied (17 cardiac explants, 2 hearts obtained at autopsy) and compared with reference hearts. Total collagen was determined by hydroxyproline analysis. Collagen types I and III were analyzed using the cyanogen bromide method and immunohistochemical analysis followed by microdensitophotometric quantification. Localization of collagen types I and III was established at the light and electron microscopic levels. Immunoelectron microscopy provided information regarding their localization. RESULTS: Total collagen and the collagen type I/type III ratio were increased in hearts with dilated cardiomyopathy (p < 0.05). Electron microscopy showed a diffuse increase in collagen fibrils in the endomysium; the perimysium showed an inhomogeneous increase. Collagen fibrils were thicker, and fibrous long-spacing collagen occurred in the endomysium. Immunoelectron microscopic findings confirmed an increase in type I collagen. CONCLUSIONS: Hearts with dilated cardiomyopathy have a statistically significant increase in the collagen type I/type III ratio. The changes occur in the endomysium and perimysium, although with differences in distribution. These changes in intramyocardial collagen may be clinically relevant because they may affect cardiac rigidity and, therefore, eventually may render the heart less compliant. Further studies are needed to evaluate at what point in the course of the disease these changes appear.

Quantification in immunohistochemistry: the measurement of the ratios of collagen types I and II.

Quantitative techniques in immunohistochemistry are needed, but they are rarely applied because of doubtful reproducibility. We have developed a method for the detection of collagen types I and III in situ. The method applied was a two-step immuno-alkaline phosphatase technique with visualization of the end-product with Fast Red. The staining intensity was measured with a microdensitometer and the results expressed as ratios. The method yielded results that were unaffected by variations in tissue section thickness but which were proportionally related to time and antigen concentrations. Leiomyoma tissue, with a ratio of collagen types I and III of approximately 1.0, was used to establish the appropriate dilutions of the antibodies, thus assuring identical optical densities. By having the leiomyoma tissue sections incubated together with the heart tissue specimens, leiomyoma tissue was also helpful in correcting deviations from the 1.0 ratio. Accurate measurements of collagen type I/III ratios in normal human heart specimens were obtained with the present quantitative immunohistochemical technique.

The neonatal heart has a relatively high content of total collagen and type I collagen, a condition that may explain the less compliant state.

OBJECTIVES: This study evaluated the extent of the collagen network in neonatal heart muscle and whether the type I/type III collagen ratio is the same as in the adult heart. BACKGROUND: The functional integrity and the stress-strain relation of heart muscle depends largely on the extracellular collagen matrix. The question therefore arises whether the altered compliance of the neonatal heart could relate to the developmental state of collagen and, in particular, the distribution of types I and III collagen. Type I collagen mainly provides rigidity and type III collagen elasticity. METHODS: Specimens from the left lateral wall of the left ventricle of human hearts (immature to full term, n = 23; 3 weeks to 12 years, n = 17) were used to determine the total collagen amount, using the hydroxyproline assay. Similar left ventricular specimens of human hearts (fetal to mature, n = 20; 2 months to 1.5 years, n = 6) were fixed in formalin, paraffin embedded and stained with Sirius red F3BA for total collagen. The ratio of total collagen to total protein was quantified spectrophotometrically. Frozen sections of left ventricular myocardium (immature to mature, n = 17; 4 months to 12 years, n = 10) were stained with antibodies raised against types I and III collagen. Antibody titration was done on human leiomyoma tissue with a known type I/type III collagen ratio. The endomysial collagen types were quantified using a spectrophotometer and expressed as a ratio. Adult human myocardium (n = 10) was used as reference. RESULTS: The study showed that the total amount of collagen increases with age. However, the ratio of total collagen to total protein and the ratio of type I to type III collagen were very high in hearts of the very young. During development, a gradual decrease occurred, with the total collagen/total protein ratio reaching normal levels at approximately 5 months after birth and the type I/type III collagen ratio stabilizing at a much later age. CONCLUSIONS: These findings suggest that the relative high content of collagen, related to the myocytes, and the high ratio of type I to type III collagen provide the substrate for a rigid, less compliant heart in neonates.

Changes in rat and mouse salivary glands and pancreas after chronic treatment with diuretics: a potential animal model for cystic fibrosis.

Defective transepithelial chloride and water transport is thought to be the cellular basis of the disease cystic fibrosis (CF). Therefore, it was attempted to develop an animal model for this disease by chronically inhibiting transepithelial chloride transport in experimental animals by long term treatment with high doses of diuretics. In the present study, changes in the salivary glands and pancreas after such treatment were investigated by X-ray microanalysis and electron microscopy. Treatment of rats for one month with diuretics caused a significant decrease in chloride and an increase in calcium in the acinar cells of the submandibular gland. This increase was due to accumulation of mucus in the cells. The strongest effect was obtained after combined treatment with furosemide and acetazolamide. Only minor changes were noted in the parotid gland and the pancreas. Treatment of mice for three months with diuretics caused similar changes in the submandibular glands. In addition, marked changes in the pancreas were observed. The chloride content of the pancreatic acinar cells was decreased. In many acinar cells, only very few zymogen granules were present. The morphological and microanalytical results point to severe dysfunction of the exocrine pancreas. These changes parallel those found in patients with CF, and the chronically furosemide-treated mouse thus could serve as an animal model for this disease.