Identification of a predominant cognitive phenotype in patients with multiple sclerosis.

BACKGROUND AND PURPOSE: Cognitive impairment occurs frequently in multiple sclerosis (MS). However, the prevalence and clinical characteristics of cognitive MS phenotype are not well established. The aim of the study was to characterize the clinical course and neurocognitive impairment of patients with MS meeting an Expanded Disability Status Scale (EDSS)-defined cognitive phenotype. METHODS: A total of 2302 patients from the Comprehensive Longitudinal Investigation of Multiple Sclerosis at Brigham and Women's Hospital (CLIMB) study were studied. Predominant cognitive MS phenotype was defined as EDSS Cerebral Functional System (FS) subscore ≥3 and remaining EDSS FS subscores ≤2 on at least one clinical visit. Demographic/clinical characteristics, phenotype stability and neurocognitive domain impairment of these subjects were assessed. RESULTS: A total of 60 of 2302 (2.6%) patients (age 52.8 ± 10.8 years, 68% female, 82% relapsing MS) met criteria for phenotype designation. A total of 29 of 60 (48%) were designated within 10 years of their presenting MS symptom. The mean cohort annualized relapse rate was 0.38 and EDSS score at last clinical assessment was 3.2 ± 1.3. Cognitive phenotype status was poorly sustained, with only 27% of subjects maintaining Cerebral FS score ≥2 throughout all follow-up. However, predominant cognitive phenotype subjects with clinical neuropsychiatric testing [n = 39/60 (65%)] frequently had cognitive impairment (1.5 SD below mean) in ≥1 domain [n = 30/39 (77%) of subjects] affecting memory, attention/executive function and processing speed. A total of 11 of 39 (28%) patients had severe-range cognitive impairment (3.0 SD below mean). Cognitive phenotype designation was associated with low rate of employment at last clinical assessment. CONCLUSION: Predominant cognitive MS phenotype is rare, although an EDSS-based definition identifies patients with multidomain cognitive impairment and may serve as a practical screen for identification of patients who might warrant close monitoring of neurocognitive status.

Comparative and developmental anatomy of cardiac lymphatics.

The role of the cardiac lymphatic system has been recently appreciated since lymphatic disturbances take part in various heart pathologies. This review presents the current knowledge about normal anatomy and structure of lymphatics and their prenatal development for a better understanding of the proper functioning of this system in relation to coronary circulation. Lymphatics of the heart consist of terminal capillaries of various diameters, capillary plexuses that drain continuously subendocardial, myocardial, and subepicardial areas, and draining (collecting) vessels that lead the lymph out of the heart. There are interspecies differences in the distribution of lymphatic capillaries, especially near the valves, as well as differences in the routes and number of draining vessels. In some species, subendocardial areas contain fewer lymphatic capillaries as compared to subepicardial parts of the heart. In all species there is at least one collector vessel draining lymph from the subepicardial plexuses and running along the anterior interventricular septum under the left auricle and further along the pulmonary trunk outside the heart and terminating in the right venous angle. The second collector assumes a different route in various species. In most mammalian species the collectors run along major branches of coronary arteries, have valves and a discontinuous layer of smooth muscle cells.

Cellular phenotypes and spatio-temporal patterns of lymphatic vessel development in embryonic mouse hearts.

BACKGROUND: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics. RESULTS: We found that scattered Lyve-1-positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox-1(+)/Lyve-1(+) cellular cords or vessels were found to invade 12.5-13.5-dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox-1(+)/Lyve-1(+) vessels were located among the Prox-1(+)/Lyve-1(-) cords and among the scattered Prox-1(-)/Lyve-1(+) cells. The Prox-1(+)/Lyve-1(-) cellular cords/tubules dominate initially at the arterial pole whereas Lyve-1(+)/Prox-1(-) cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve-1(+)/CD45(+), Lyve-1(+)/CD11b(+), Lyve-1(+)/F4/80(+) and Lyve-1(+)/CD68(+) cells were subsequently found to be co-opted to the wall of the developing lymphatic vessels while gaining Flk-1. CONCLUSIONS: Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart.

Diversity of coronary arterial tree in laboratory mice.

BACKGROUND: Research on the development and topography of mouse coronary arteries has been conducted for many years. Patterns of the course of these vessels have been described in various mouse strains. Our research focused on hearts of MIZZ mice. MATERIALS AND METHODS: We visualised the coronary artery system by means of latex dye perfusion via the aorta. The injected latex did not reach the capillary vessel system. RESULTS: The heart of MIZZ mice is supplied with blood by two main coronary arteries: the right and the left one. They deliver blood to the right and left part of the heart, respectively. The right coronary artery arises from the right sinus of the aorta and the left coronary artery from the left sinus. The interventricular septum is usually supplied by the septal artery, which is the main branch of the right coronary artery. All arteries of the coronary system run intramurally. The number of branches and the location of their ostia differed among the examined individuals. CONCLUSIONS: Detailed information about the normal topography of coronary arteries, the number and course of their branches, as well as the area of the heart which is vascularised by these vessels constitutes the basic knowledge necessary to conduct further experiments.

The Correlation of PPARα Activity and Cardiomyocyte Metabolism and Structure in Idiopathic Dilated Cardiomyopathy during Heart Failure Progression.

This study aimed to define relationship between PPARα expression and metabolic-structural characteristics during HF progression in hearts with DCM phenotype. Tissue endomyocardial biopsy samples divided into three groups according to LVEF ((I) 45-50%, n = 10; (II) 30-40%, n = 15; (III) <30%, n = 15; and control (donor hearts, >60%, n = 6)) were investigated. The PPARα mRNA expression in the failing hearts was low in Group (I), high in Group (II), and comparable to that of the control in Group (III). There were analogous changes in the expression of FAT/CD36 and CPT-1 mRNA in contrast to continuous overexpression of GLUT-4 mRNA and significant increase of PDK-4 mRNA in Group (II). In addition, significant structural changes of cardiomyocytes with glycogen accumulation were accompanied by increased expression of PPARα. For the entire study population with HF levels of FAT/CD36 mRNA showed a strong tendency of negative correlation with LVEF. In conclusion, PPARα elevated levels may be a direct cause of adverse remodeling, both metabolic and structural. Thus, there is limited time window for therapy modulating cardiac metabolism and protecting cardiomyocyte structure in failing heart.

Angiotensin II associated cardiac myocyte necrosis: role of adrenal catecholamines.

OBJECTIVE: Cardiac myocyte necrosis is associated with an endogenous increase in angiotensin II or a subpressor dose of angiotensin II. In each case, raised plasma angiotensin II, together with aldosterone, results in cardiac myocyte necrosis followed by scarring. Arterial hypertension is non-contributory. The aim of this study was to determine whether myocyte necrosis is induced directly by angiotensin II, or indirectly by catecholamines or aldosterone released from adrenal glands in response to angiotensin II. METHODS: Right and left ventricular myocardium was examined over a two week period in rats receiving a subpressor yet cardiotoxic dose of angiotensin II (150 ng.min-1.kg-1 subcutaneously) alone or together with total adrenalectomy, bilateral medullectomy, or the aldosterone receptor antagonist spironolactone (150-200 mg.d-1.kg-1). Findings were compared to unoperated age/sex matched controls. Coronal sections obtained from the base, equator and apex of the heart were stained with antifibronectin antibody to detect myocyte injury, picrosirius red for fibrillar collagen, or haematoxylin and eosin. RESULTS: In both right and left ventricles (a) fibronectin labelling was distributed within injured cardiac myocytes and adjacent interstitium of both ventricles in rats receiving angiotensin II or angiotensin II plus spironolactone; (b) fibronectin labelling of myocytes was markedly diminished or absent in the ventricles of rats with total adrenalectomy or bilateral medullectomy; and (c) there was microscopic scarring of both ventricles after two weeks of either angiotensin II or angiotensin II plus spironolactone, but not in medullectomised or adrenalectomised animals. CONCLUSIONS: In the presence of increased circulating levels of angiotensin II, bilateral medullectomy and total adrenalectomy (in contrast to spironolactone) reduced myocyte injury and scarring of the right and left ventricular myocardium, suggesting that the cytotoxic effect of angiotensin II is largely indirect and related to circulating catecholamines released by the adrenal medulla.

Coronary vascularization during development in the rat and its relationship to basic fibroblast growth factor.

OBJECTIVE: Our overall aims were to elucidate the temporal and spatial sequence of coronary vascularization during development in the rat, and to determine whether basic fibroblast growth factor expression corresponds to any phase of the vascularization process. METHODS: Immunohistochemical, histochemical, morphometric and in situ hybridization analyses were performed on prenatal and postnatal hearts of various ages. RESULTS: Coronary vascularization, which begins at embryonic day 13 (E13) with blood island-like structures in the epicardium, progresses from this layer toward the endocardium as indicated by a transmural gradient of vascular volume throughout the ventricles. Vascular smooth muscle first appears in E17 hearts at the time a capillary-like plexus coalesces and penetrates the aorta to form the main coronary arteries. These vessels maintain an anastomatic morphology and must undergo subsequent remodeling in order to assume adult branching characteristics. The early postnatal period is characterized by development of the arterial tree and the enzymatic differentiation of the arteriolar and venular ends of the capillary bed. Although bFGF is expressed both prenatally and postnatally, the highest mRNA expression was noted during the early period of vascularization (E14 and E15), and the early neonatal period (1-6 days) which corresponds to a period of substantial microvascular growth. CONCLUSIONS: Coronary vascularization follows a temporal sequence which includes transmural expansion of the capillary bed, arteriolar formation subsequent to vascular penetration of the aorta, and postnatal growth, differentiation, and remodeling. Since high levels of bFGF expression are correlated with key time points in coronary vascular growth, bFGF may play an important role in this process.

Angiotensin converting enzyme and kininase-II-like activities in cultured valvular interstitial cells of the rat heart.

OBJECTIVE: The function of angiotensin converting enzyme (ACE) at cell sites of high collagen turnover, such as heart valves, is uncertain. The aim of this study was to assess ACE and kininase-II-like activities and collagen turnover in cultured valvular interstitial cells of the adult rat heart. METHODS: The valvular interstitial cell phenotype was determined by immunolabelling (rhodamine phalloidin, desmin, and Griffonia simplicifolia lectin), and the presence of ACE mRNA and protein was confirmed by reverse transcriptase-polymerase chain reaction analysis, ACE monoclonal antibody and in vitro autoradiography, respectively. ACE and kininase-II-like activities in valvular interstitial cells were analysed by high performance liquid chromatography. Angiotensin II (AT1) and bradykinin receptors in valvular interstitial cell membranes were examined by western immunoblotting and binding assay. Type I collagen and collagenase in valvular interstitial cell culture media were determined by ELISA and zymography, respectively. Type I collagen mRNA expression in cultured valvular interstitial cells was determined by northern blot analysis and in situ hybridisation. RESULTS: In intact valvular interstitial cells or their cell membrane we found: (1) actin microfilaments, but not desmin or lectin labelling; (2) ACE mRNA expression and binding activity; (3) conversion of angiotensin I to angiotensin II, which was completely inhibited by 50 microM lisinopril, while kinase-II-like activity exceeded ACE activity and was not inhibited by lisinopril; (4) AT1 and bradykinin receptors in valvular interstitial cell membrane preparations; (5) type I collagen mRNA expression and collagenase activity; and (6) angiotensin II induced increase in type I collagen synthesis and mRNA expression. CONCLUSIONS: Cultured valvular interstitial cells represent a nonendothelial, non-smooth-muscle cell type that expresses mRNA for ACE and type I collagen. ACE and kininase-II-like activities in valvular interstitial cells may be involved in the regulation of peptides that influence collagen turnover. Angiotensin II stimulates type I collagen synthesis and mRNA expression in these cells.

Fibronectin accumulation within cardiac myocytes in rats with elevated plasma angiotensin II.

Elevation in plasma angiotensin II (AngII) is associated with cardiac myocyte necrosis. Myocyte necrosis followed by wound healing and fibrosis represents a structural remodeling of the myocardium thought to contribute to abnormal myocardial function. Fibronectin (FN) is generally considered an early component of the healing process that precedes collagen accumulation. To better understand the time course of this remodeling process involving both cardiac myocytes and extracellular matrix, (i.e., FN and collagen), we used two animal models: (1) endogenous activation of the renin-angiotensin system by surgical induction of renovascular hypertension and (2) exogenous AngII administration (150 ng/min/kg). Animals were killed at different time points within the first two weeks. Both "cellular" (cFN) and "plasma" (pFN) FN immunolabeling were compared with collagen distribution (picrosirius red stain), together with histopathologic (hematoxylin-eosin stain) and ultrastructural examination of cardiac myocytes. In each experimental group, the pattern and time course of FN immunolabeling was coincident with histopathologic evidence of myocyte injury and/or remodeling. We found different patterns of FN labeling of cardiac myocytes: (a) homogenous intracellular distribution in necrotic myocytes, most obvious on days 1 and 2; (b) patchy intracellular distribution in nonnecrotic myocytes starting on day 4; and (c) marking internalized capillaries. Both FNs were codistributed throughout the myocardium of each ventricle; however, cFN was less pronounced and not seen in mature scars. Ultrastructural examination revealed different kinds of intramyocytic inclusions, characterized by vacuoles containing fibrillar/flocculent material, remnants of unknown origin, or internalized capillaries. We conclude that FNs are markers of cardiac myocyte necrosis and early interstitial remodeling and that renovascular hypertension and AngII administration exhibit the same time course and pattern of FN and collagen expression.

Prenatal development of coronary arteries in the rat: morphologic patterns.

The aim of this work was to address spatiotemporal and morphologic patterns of coronary artery development in rats, based on immunohistochemical and ultrastructural studies of hearts at different stages of prenatal development. Griffonia simplicifolia I lectin and alpha-smooth muscle antibody were used to demonstrate endothelial cells and/or their precursors and smooth muscle cells, respectively. Ultrastructural examination was performed on ED14-16 hearts to study the morphology of the developing coronary arteries in different regions of the truncus arteriosus and adjacent myocardium. On ED14 endothelial-like cells present within the mesenchyme surrounding the outflow tract penetrated the aortic wall and the truncoconal proximal myocardium. On ED15 these penetrating cells formed vascular clusters, which were the first signs of presumptive vascular channels. Development of the coronary artery proceeded by coalescence of discontinous vascular clusters, formation of the lumen (vascular channels) and establishing a connection of the proximal part with the aorta. The second layer of cells around vascular channels (embryonic media) consisted of mesenchymal cells that were attracted to the immature vessel and were first seen on ED15. At this time no lumenized connection of the coronary artery with the aorta has been seen. After the lumenized connection of the coronary artery with the aorta had been established perivascular cells of the media started to differentiate into vascular smooth muscle, as was shown by alpha-smooth muscle actin-staining. Further development and differentiation of the media and adventitia proceeded distally (towards the apex).

Prenatal development of coronary arteries in the rat: morphometric patterns.

The aim of this work was to address morphometric patterns of coronary artery (c.a.) development in the rat based on serial section analysis of hearts at different stages of prenatal development. Studies were performed on foetal hearts 15-21 days (ED) post-conception. Paraffin sections were stained with haematoxylin-eosin (H&E) and frozen sections were labelled with Griffonia simplicifolia I (GSI) lectin (endothelial cell marker). Coronary arteries' luminal diameters were measured at different distances from the aortic roots and the main c.a. branch lengths were calculated from serial sections. All measured values were compared to heart length and to foetal stages. On ED15 precursors of c.a. were distinguished as tubes running on both sides of the outflow tract. Below the aortic valves the tubes had the largest diameter. Formation and development of c.a. proceeded by elongation of vascular tubes distally, ramification and formation of the media and the adventitia. During the prenatal period the c.a. length increased approximately 14-fold, while heart length increased about 4-fold, and crown-rump length about 2.5-fold. The lumen of the proximal part of c.a. increased 4-fold during ED18-21. An increase in c.a. length is the fastest compared to the heart growth, and crown-rump growth during the foetal life.

Vasculogenesis of the embryonic heart: contribution of nucleated red blood cells to early vascular structures.

During embryogenesis, coronary vessels develop via vasculogenesis and angiogenesis. Vasculogenesis is formation in situ of primary vessels from angioblasts - endothelial cell progenitors, and angiogenesis is formation of vessels from the existing ones. In the embryonic heart vasculogenesis precedes and overlaps angiogenesis and lasts till the end of the fetal life. What is unique about heart vasculogenesis is the fact that nucleated blood cells accompany early angioblasts in a spatiotemporal way. Morphologically these structures resemble yolk sac blood islands, thus, they have been called blood-island-like structures. In addition, these early vascular structures (blood-island-like) are found in the heart before coronary vessel system connects with the systemic circulation. We present the recent data regarding endothelial cell properties and derivation during coronary vessel formation and hypotheses concerning a source of blood cells in early vascular structures of the heart; the latter has received little attention in the literature. This review summarizes current knowledge on the endothelial cell origination from epicardial mesothelium or liver primordium. This review also focuses on blood cell contribution to coronary vessel vasculogenesis. The role of proepicardium in the epicardial cover formation and the epicardium as a source of cellular components of coronary vasculature and interstitial fibroblasts is presented. It seems that blood cells and angioblasts, which form the early vascular structures do not derive from the same hemangioblastic precursor.

Some extracellular matrix elements as markers of 'capillary tunnels' in hypertrophied rat heart.

We studied the distribution of the extracellular matrix proteins fibronectin (FN) and laminin (LM) in the hypertrophied hearts of spontaneously hypertensive rats (SHR), using an immunofluorescence method with specific antibodies. The immunohistochemical reaction was positive in the cytoplasm of some hypertrophied cardiomyocytes. The results showed that FN and LM can be used as markers for tunnels, i.e. intracardiocytic invaginations of the sarcolemma. The tunnels observed contained capillaries.

Bradykinin receptor and tissue ACE binding in myocardial fibrosis: response to chronic angiotensin II or aldosterone administration in rats.

High density angiotensin converting enzyme (ACE) binding is present in the perivascular fibrosis involving intramyocardial coronary arteries and the microscopic scarring of the myocardium that accompanies chronic elevations in circulating angiotensin II (AngII) and/or aldosterone (ALDO). As a kininase II, ACE degrades bradykinin. Herein we sought to determine whether bradykinin (BK) receptor binding was associated with ACE binding in each of these experimental models. BK receptor binding was localized and quantified by in vitro quantitative autoradiography, using [125I-Tyr8]BK. In serial sections of the same heart hematoxylin and eosin (H&E) and picrosirius red (PSR) staining were utilized to address cardiac myocyte injury and fibrosis, respectively. Four experimental groups were examined: unoperated, untreated, age/sex matched controls: age/sex matched uninephrectomized control rats receiving a high sodium diet; animals that received AngII (9 micrograms/h sc) for 2, 4 or 6 weeks; and uninephrectomized rats on a high sodium diet that received ALDO (0.75 micrograms/h sc) for similar periods of time. We found: (a) myocardial fibrosis, including perivascular fibrosis and microscopic scarring, at week 2 of AngII, but not until week 4 or more of ALDO treatment; (b) low BK receptor binding in normal ventricles that was increased in scars and markedly increased in perivascular fibrosis at week 2 of AngII and each increased further at week 4 and 6 of AngII: (c) low BK receptor binding at week 2 and 4 weeks of ALDO treatment which became markedly increased at fibrous tissue sites at week 6. BK receptor and ACE binding were anatomically coincident and localized to each site of fibrosis in both models. The co-location of BK receptor and ACE binding in these models raises the prospect that fibrous tissue ACE may utilize BK as substrate and BK, in turn, may play a role in fibrous tissue formation.

Inhibition of angiotensin-converting enzyme and attenuation of myocardial fibrosis by lisinopril in rats receiving angiotensin II.

High-density angiotensin-converting enzyme (ACE) binding is present in heart valve leaflets and the fibrous tissue that appears in the rat myocardium after either chronic administration of angiotensin II (AngII) or after myocardial infarction. This suggests that connective tissue ACE is independent of circulating AngII and that ACE may be an integral component of normal and pathologic tissue repair. To address this possibility the present study was undertaken. We sought to determine whether the ACE inhibitor lisinopril would attenuate fibrous tissue ACE binding and fibrous tissue formation in the myocardium of rats receiving AngII. Three experimental groups were examined: untreated, age-matched controls; rats receiving subcutaneous AngII (150 ng/min) by minipump for 2 weeks; and rats receiving AngII plus oral lisinopril (20 mg/kg/day) for 2 weeks. Cardiac ACE binding density was localized and quantified by in vitro autoradiography with [125I]-labeled 351A, a tyrosyl derivative of lisinopril, while fibrosis was identified by light microscopy in serial sections stained with picrosirius red. Hematoxylin and eosin and anti-fibronectin antibody were used to identify cardiac myocyte necrosis. Immunohistochemical labeling with alpha-smooth muscle actin was used to identify myofibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of the smooth muscle cell phenotypes during embryonic development of coronary vessels in the rat.

Smooth muscle cell (SMC) maturation during embryonic development of coronary arteries and veins was studied in rats using different markers of the contractile phenotypes. The spatio-temporal pattern of distribution of these markers compared with the developing tunica media was examined. Alpha-smooth muscle actin (alpha-SMA) was the first marker of the SMC in the tunica media of coronary arteries found in ED16 hearts, followed by smooth muscle myosin heavy chain isoform which occurred on ED17. Subsequently 1E12 antigen was expressed in coronary artery wall in ED18 hearts, and finally smoothelin. The markers occur within the proximal part of the coronary arteries and deploy toward the apex. They are also found within the great vessels. None of the markers except for the alpha-SMA were found in coronary veins during embryonic life. We conclude that the SMC population of the developing tunica media of coronary vessels differentiates by the acquisition of particular markers and this process lasts till the end of the prenatal and early postnatal life.

Angiotensin-converting enzyme and myocardial fibrosis in the rat receiving angiotensin II or aldosterone.

Angiotensin-converting enzyme (ACE) is present in tissues composed largely of fibrillar collagen such as heart valves, the adventitia of great vessels and intramyocardial coronary arteries, and the scar that follows myocardial infarction. We tested the hypothesis that such tissue ACE is related to fibrous tissue formation and that its appearance is independent of circulating renin and angiotensin peptides. For this purpose we selected experimental models that simulate primary and secondary hyperaldosteronism, each of which are associated with the appearance of myocardial fibrosis. ACE in the excised heart and aorta was localized by in vitro autoradiography with [125I]351A, while fibrosis was identified by light microscopy in sections stained with collagen specific picrosirius red. Tissue was obtained at 2, 4, and 6 weeks from various experimental groups: unoperated, untreated, age- and sex-matched controls; age- and sex-matched uninephrectomized control rats on a high sodium diet; and rats that had received either aldosterone (ALDO) or angiotensin II (AII). Compared with controls, we found ACE binding (1) unchanged after 2 weeks of ALDO, but increased in the adventitia of intramural coronary arteries after 4 weeks, in keeping with the perivascular fibrosis that appeared in each ventricle; (2) markedly increased after 6 weeks of ALDO, where it not only involved coronary vessels but also microscopic scars that appeared in atria and ventricles; (3) increased in the coronary adventitia and sites of scarring, each of which were present 2 weeks after AII; and (4) markedly increased after 4 and 6 weeks of AII as fibrosis became more extensive.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial matrix metalloproteinase(s): localization and activation.

Matrix metalloproteinases (MMPs) and neutrophil elastase (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37 degrees C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5 +/- 0.2 x 10(5) M-1min-1 and for oxidized glutathione (GSSG) 1.5 +/- 0.1 M-1min-1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.

Tunnel capillaries in hypertrophied myocardium of rats with aorto-caval fistula.

Male inbred rats (Wag) with aorto-caval fistula were used as a model of volume-overloaded heart hypertrophy. Hearts were taken after 1, 2, and 6 months postoperatively for immunohistochemical, histological and ultrastructural investigations. Immunohistochemical staining with some anti-extracellular matrix antibodies and ultrastructural findings allowed us to recognize intracardiocytic tunnels which were intracellular invaginations of plasmalemma surrounded by basement membrane. Inside the tunnels endothelial cells forming well-developed capillaries were entrapped. Tunnel capillary formation is discussed as an adaptive response to increased cardiac work due to volume overload.