Isolation, characterization, and localization of cardiac collagen type VI. Associations with other extracellular matrix components.

We have isolated and characterized collagen type VI from murine, canine, and nonhuman primate hearts. In the three species studied, collagen type I was the major collagenous component of the cardiac interstitium (80% of total collagen), whereas collagen type VI represented approximately 5% of total collagen. To define the exact distribution of collagen type VI and its possible interactions with other components of the cardiac extracellular matrix, collagen types I, III, IV, and VI, laminin, and fibronectin were localized in the rat myocardium by immunohistochemistry, using monospecific antibodies. In the rat myocardium, collagen type VI was prevalent in the media and adventitia of muscular arteries, in fine connective tissue septa, in the area surrounding capillaries, and in the delicate endomysium in proximity to myocardial cells. When compared with the immunohistochemical localization of collagen types I, III, and IV, laminin, and fibronectin, the continuity and hierarchical organization of the cardiac extracellular matrix became apparent. The matrix forms a continuous network extending from the pericardium to the endocardium. Furthermore, there is an arborescent hierarchy in the system such that collagen type I is more prevalent in the wider septa, collagen type III being more obvious in medium-sized branches, and fibronectin and collagen type VI prevailing in the terminal (pericellular) aspects of the network. In this pericellular location, fibronectin and collagen type VI, by means of specific interactions, may act as anchor components linking the myocardial cell basement membranes not only to the extracellular matrix but also to the cardiac interstitial cells. This continuity, organization, and coupling of the cardiac extracellular matrix appears well suited to integrate and distribute the physical stress generated by the continuous contraction and relaxation of the myocardium.     

Origin of mesenchymal tissue in the septum primum: a structural and ultrastructural study

A structural, ultrastructural and histochemical study in chick embryos indicates that the septum primum mesenchymal tissue originate between 3 and 5 days of development and that their origin may be related to an activation of endocardial cells that cover the septum primum. By day 3, endocardial cells display migratory appendages, cell hypertrophy and an increase in secretory and mitotic activity. In later stages (day 4) hypertrophic endocardial cells undergoing division seem to delaminate and translocate toward the subendocardial space to give rise to free mesenchymal-type cells. These results suggest that the endocardium makes up the bulk of the septum primum mesenchymal tissue as has been demonstrated during mesenchymal tissue formation in the atrioventricular canal and outflow tract. Before and during mesenchymal tissue formation an accumulation of extracellular matrix components like proteoglycans can be visualized using tannic acid. These extracellular components might be related to the promotion of cellular events described during endocardial activation. The fusion of the septum primum with the atrioventricular (AV) endocardial cushions which would obliterate the foramen primum, occurs between mesenchymal tissues. Therefore, any alteration in the normal development of these mesenchymal tissues could be related to pathological cases of persistent atrial communications. Light microscopy preliminary observations of embryonic mouse heart indicate that septum primum mesenchymal tissue formation occurs similarly between mouse and chick embryos.     

Matrix modulation and heart failure: new concepts question old beliefs

PURPOSE OF REVIEW: Myocardial remodeling is a complex process involving several molecular and cellular factors. Extracellular matrix has been implicated in the remodeling process. Historically, the myocardial extracellular matrix was thought to serve solely as a means to align cells and provide structure to the tissue. Although this is one of its important functions, evidence suggests that the extracellular matrix plays a complex and divergent role in influencing cell behavior. This paper characterizes some of the notable studies on this dynamic entity and on adverse myocardial remodeling that have been published over the past year, which further question the belief that the extracellular matrix is a static structure. RECENT FINDINGS: Progress has been made in understanding how the extracellular matrix is operative in the three major conditions (myocardial infarction, left ventricular hypertrophy due to overload, and dilated cardiomyopathy) that involve myocardial remodeling. Several studies have examined plasma profiles of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases following myocardial infarction and during left ventricular hypertrophy as surrogate markers of remodeling/remodeled myocardium. It has been demonstrated that bioactive signaling molecules and growth factors, proteases, and structural proteins influence cell-matrix interactions in the context of left ventricular hypertrophy. Finally, studies that either removed or added tissue inhibitor of metalloproteinases species in the myocardium demonstrated the importance of this regulatory protein in the remodeling process. SUMMARY: Understanding the cellular and molecular triggers that in turn give rise to changes in the extracellular matrix could provide opportunities to modify the remodeling process.     

Distribution of the neural cell adhesion molecule (NCAM) during heart development

The neural cell adhesion molecule, NCAM, was localized in the embryonic chick heart from Hamburger-Hamilton stage 14 up to hatching and in the adult heart. A monoclonal antibody directed to NCAM was used with the indirect antibody technique to stain frozen sections with immunoperoxidase. The myocardium showed immunoreactivity at stages 15 and 21, with little to no staining of epicardium, endocardium or atrioventricular endocardial cushion tissue. At stage 22, additional immunoreactivity was found in the endocardium of both the atrial septum and the atrial and ventricular surfaces of the atrioventricular cushions. Endocardial-derived mesenchymal cells within the cushions were also immunostained for NCAM. A gradient of NCAM staining was evident in the ventricular wall by stage 16. The staining intensity in the myocardium subjacent to the epicardium was less than found near the ventricular lumen. Biochemical analyses revealed that the embryonic heart expresses polysialylated NCAM. Upon desialylation with the endoneuraminidase Endo-N, the predominant heart NCAM has an apparent molecular weight of 155 to 160 kDa, which is distinct in size from the predominant forms found in embryonic chick nervous system (180, 140 and 120 kDa). NCAM expression is regionally regulated in the heart. The pattern of its expression is consistent with our hypothesis that it is involved in (1) differentiation of the atrial and ventricular walls, (2) fusion of the atrial septum with the endocardial cushions, (3) fusion of the endocardial cushions, and (4) formation and remodeling of ventricular trabeculae.     

Alpha8beta1 integrin is upregulated in myofibroblasts of fibrotic and scarring myocardium

Integrins mediate cell attachment to the extracellular matrix (ECM) regulating migration, proliferation, and differentiation. We previously reported the presence of alpha8beta1 integrin on cultured cardiac fibroblasts. Extending this information, we localized alpha8beta1 integrin in normal rat myocardial tissue, and investigated its expression pattern in rats chronically infused with angiotensin II (Ang II, 500 ng/kg/min), a well-recognized profibrotic factor. Alpha8beta1-integrin expression was analyzed by binding assay, western blotting, and immunohistochemistry. In normal myocardium, immunohistochemical staining for alpha8 was found in fibroblasts, as well as in the epicardium, endocardium, and valves. Vascular smooth muscle cells (VSMCs) of the media of cardiac arteries also stained positively. After 14-d-Ang II infusion, staining for fibronectin, as well as collagen staining by Sirius red, revealed extensive interstitial and perivascular fibrosis. Increased expression of alpha8 integrin in ventricular smooth muscle (SM) alpha-actin-positive fibroblasts (myofibroblasts) was also recorded. The upregulation of alpha8beta1 integrin was confirmed by binding assay and by western blotting. Microscopic scars, a characteristic of reparative fibrosis, were invaded by matrix proteins and by strongly alpha8- and SM alpha-actin-positive myofibroblasts. The results indicate that, in rat adult myocardium, alpha8beta1 integrin is expressed in fibroblasts and VSMC. In Ang II-infused animals, alpha8beta1-integrin expression was enhanced in the left ventricle and arteries. The coordinate regulation of alpha8beta1 integrin on fibroblasts and ECM proteins raises the possibility that this integrin is implicated in the deposition of matrix components leading to fibrosis.     

Accessory atrioventricular node with properties of a typical accessory pathway: anatomic-electrophysiologic correlation

We report an accessory AV node producing ventricular preexcitation and comprising the retrograde limb of AV reentrant tachycardia (AVRT). A 66-year-old man presented with an anteroseptal myocardial infarction and thereafter developed recurrent, drug-refractory AVRT requiring multiple cardioversions. Electrophysiologic findings were typical for a concealed anteroseptal accessory pathway 0.5 cm anterior to the His bundle. The patient died of intractable heart failure after endocardial resection for a left ventricular aneurysm and coronary bypass grafting. Pathologic examination revealed a para-Hisian accessory AV node connecting the right atrium to ventricular myocardium immediately anterior to the His bundle at a depth of 4 mm from the endocardium. No typical AV accessory pathway was found. This is the first report of an accessory AV node that participated in AVRT. It was deeper than typical radiofrequency catheter ablation lesions.     

Effects of beta-aminopropionitrile fumurate (BAPN) on early heart development

Collagen is a major constituent of the extracellular matrix (ECM) of the early developing heart. The role of mature cross-linked collagen has not been demonstrated in cardiac morphogenesis, although investigators using other developmental systems have suggested that collagen can act as an inducer. The injection of 500 μg of BAPN (beta-aminopropionitrile fumurate), a known inhibitor of cross-linking, into the pericardial region of chick embryos prior to valvular and septal morphogenesis (Stage 12 to 14) was without effect on the appearance or migration of cushion tissue mesenchyme in the ECM. However, by Stage 22 to 23 BAPN-treated embryos exhibited truncal aneurysms which resulted in death. Ultrastructural examination of the hearts of BAPN-treated embryos showed an alteration in the cell surface of both the endocardium and myocardium as evidenced by the deposition of very electron dense material. Biochemical analysis confirmed that BAPN-treated organisms were indeed lathyritic. These studies suggest that the continued synthesis and deposition of mature cross-linked collagen is not required for initiation and migration of cushion tissue mesenchyme nor for the appearance and maintenance of specific endocardial shape changes referred to as “flutes”. It is suggested that collagen is primarily a structural macromolecule in the early heart, rather than an inducer. These studies further suggest that, in addition to its effect on collagen cross-linking, BAPN may cause cardiac anomalies by modifying collagen: proteoglycan interaction within the ECM.     

Hyperplastic conotruncal endocardial cushions and transposition of great arteries in perlecan-null mice

Perlecan is a heparan-sulfate proteoglycan abundantly expressed in pericellular matrices and basement membranes during development. Inactivation of the perlecan gene in mice is lethal at two developmental stages: around E10 and around birth. We report a high incidence of malformations of the cardiac outflow tract in perlecan-deficient embryos. Complete transposition of great arteries was diagnosed in 11 out of 15 late embryos studied (73%). Three of these 11 embryos also showed malformations of semilunar valves. Mesenchymal cells in the outflow tract were abnormally abundant in mutant embryos by E9.5, when the endocardial-mesenchymal transformation starts in wild-type embryos. At E10.5, mutant embryos lacked well-defined spiral endocardial ridges, and the excess of mesenchymal cells obstructed sometimes the outflow tract lumen. Most of this anomalous mesenchyme expressed the smooth muscle cell-specific alpha-actin isoform, a marker of the neural crest in the outflow tract of the mouse. In wild-type embryos, perlecan is present in the basal surface of myocardium and endocardium, as well as surrounding presumptive neural crest cells. We suggest that the excess of mesenchyme at the earlier stages of conotruncal development precludes the formation of the spiral ridges and the rotation of the septation complex in order to achieve a concordant ventriculoarterial connection. The observed mesenchymal overpopulation might be due to an uncontrolled migration of neural crest cells, which would arrive prematurely to the heart. Thus, perlecan is involved in the control of the outflow tract mesenchymal population size, underscoring the importance of the extracellular matrix in cardiac morphogenesis.     

Maintenance of radiation-induced intestinal fibrosis： Cellular and molecular features

Recent advances in cell and molecular radiobiology clearly showed that tissue response to radiation injury cannot be restricted to a simple cell-killing process, but depends upon continuous and integrated pathogenic processes, involving cell differentiation and crosstalk between the various cellular components of the tissue within the extracellular matrix. Thus, the prior concept of primary cell target in which a single-cell type (whatever it’s epithelial or endothelial cells) dictates the whole tissue response to radiation injury has to be replaced by the occurrence of coordinated multicellular response that may either lead to tissue recovery or to sequel development. In this context, the present review will focus on the maintenance of the radiation-induced wound healing and fi brogenic signals triggered by and through the microenvironment toward the mesenchymal cell compartment, and will highlight how sequential and sustained modifi cations in cell phenotypes will in cascade modify cell-to-cell interactions and tissue composition.     

Extracellular Matrix and Growth Factors During Heart Growth

The effects of growth factors on tissue remodeling and cell differentiation depend on the nature of the extracellular matrix, the type and organization of integrins, the activation of metalloproteinases and the presence of secreted proteins associated to the matrix. These interactions are actually poorly known in the cardiovascular system. We describe here: 1) the main components of extracellular matrix within the cardiovascular system; 2) the role of integrins in the transmission of growth signals; 3) the shift in the expression of the components of the extracellular matrix (fibronectin and collagens) and the stimulation of the synthesis of metalloproteinases during normal and hypertrophic growth of the myocardium; 4) the effects of growth factors, such as Angiotensin II, Fibroblast Growth Factors (FGF), Transforming Growth Factor- (TGF-), on the synthesis of proteins of the extracellular matrix in the heart.     

Lineage and morphogenetic analysis of the cardiac valves

We used a genetic lineage-labeling system to establish the material contributions of the progeny of 3 specific cell types to the cardiac valves. Thus, we labeled irreversibly the myocardial (alphaMHC-Cre+), endocardial (Tie2-Cre+), and neural crest (Wnt1-Cre+) cells during development and assessed their eventual contribution to the definitive valvar complexes. The leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity, and the leaflets of the outflow tract valves were all found to be generated from mesenchyme derived from the endocardium, with no substantial contribution from cells of the myocardial and neural crest lineages. Analysis of chicken-quail chimeras revealed absence of any substantial contribution from proepicardially derived cells. Molecular and morphogenetic analysis revealed several new aspects of atrioventricular valvar formation. Marked similarities are seen during the formation of the mural leaflets of the mitral and tricuspid valves. These leaflets form by protrusion and growth of a sheet of atrioventricular myocardium into the ventricular lumen, with subsequent formation of valvar mesenchyme on its surface rather than by delamination of lateral cushions from the ventricular myocardial wall. The myocardial layer is subsequently removed by the process of apoptosis. In contrast, the aortic leaflet of the mitral valve, the septal leaflet of the tricuspid valve, and the atrioventricular fibrous continuity between these valves develop from the mesenchyme of the inferior and superior atrioventricular cushions. The tricuspid septal leaflet then delaminates from the muscular ventricular septum late in development.     

The Extracellular Matrix and the Cytoskeleton in Heart Hypertrophy and Failure

Cell characteristics and phenotype depend on the nature of the extracellular matrix, the type and organization of integrins and cytoskeleton. The interactions between these components are poorly known at the myocyte level and during cardiac remodeling associated with cardiac hypertrophy and heart failure. We analyze here the nature and organization of extracellular matrix (ECM) proteins, cytoskeleton and integrins and their regulation by growth factors, such as angiotensin II, in normal myocyte growth and in pathological growth (hypertrophy) of the myocardium and heart failure.     

The relationship between ventricular arrhythmias and ischemia-induced conduction delay in closed-chest animals within 24 hours of myocardial infarction

To investigate the myocardial conduction characteristics of premature impulses during the first 24 hours following coronary ligation and its relationship to late infarction ventricular arrhythmias, transmural electrodes were positioned in the normal and ischemic myocardium in nine dogs. Cardiac conduction in ischemic myocardium was delayed 15 minutes post coronary occlusion both in the epicardium and endocardium, both in the anterograde (base to apex) and retrograde (apex to base) direction, and was maintained at the same level throughout the experiment. Conduction across the border of ischemic myocardium from ischemic to the normal segment was also delayed, especially in the endocardium. Spontaneous ventricular arrhythmias recorded on Holter tapes showed significant increase in the number of premature ventricular complexes and ventricular tachyarrhythmias 9 hours after infarction. Thus our findings suggest that spontaneous arrhythmias occurring in the late phase of acute myocardial infarction (AMI) are independent of the ischemia-induced conduction delay and an alternate mechanism such as abnormal automaticity may be responsible for late ventricular arrhythmias.     

Effects of carvedilol on cardiac cytokines expression and remodeling in rat with acute myocardial infarction

OBJECTIVE: A number of observations suggest that cytokines may be important modulators in the ventricular remodeling process. It is unclear whether carvedilol modulates myocardial pro-inflammatory and anti-inflammatory cytokines expression. We hypothesized that carvedilol could improve ventricular remodeling partly through the modulation of cytokines. The goal of this study was to evaluate the effects of carvedilol on cardiac cytokines expression as well as on myocardial and extracellular matrix remodeling in rats with acute myocardial infarction. METHODS: Rats with AMI induced by left anterior descending branch ligation were randomized to carvedilol and control group which were further compared to sham-operated group. We studied the effects of 4-weeks therapy with carvedilol starting 24 h after infarction on 1) hemodynamics, 2) tissue weights, 3) myocardial cytokines (TNF-alpha, IL-1beta, IL-6, IL-10 and TGF-beta1) expression by semi-quantitative RT-PCR and immunoblotting, 4) matrix metalloproteinases activity by gelatin zymography, 5) collagen expression by immunohistochemistry, 6) myocardium fetal gene (alpha and beta myosin heavy chain) expression. RESULTS: Treatment with carvedilol 1) reduced the pro-inflammatory cytokines and fibrogenic cytokine TGF-beta1 levels in myocardium and was associated with the amelioration of the elevated left ventricular diastolic pressure. 2) increased anti-inflammatory cytokine, IL-10 protein expression. 3) reduced matrix metalloproteinases-2 and matrix metalloproteinases-9 activity 4) reduced myocardial collagens 5) did not modify fetal gene re-expression. CONCLUSION: Pro-inflammatory, anti-inflammatory and fibrogenic cytokines are all involved in the process of post-infarction myocardial remodeling. One mechanism underlying the beneficial effects of carvedilol on post-infarction myocardial remodeling may be modulation of the balance between pro- and anti-inflammatory cytokines as well as fibrogenic cytokines and extracellular matrix (ECM) remodeling.     

经心内膜心肌内移植猪自体骨髓间充质干细胞治疗心肌梗死的实验方法

目的:探讨经皮经心内膜心肌注射移植猪自体骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的治疗心肌梗死(MI)实验方法的可行性和安全性。方法: 选5只雌性健康小型猪通过股动脉入路采用冠脉球囊封堵冠状动脉左前降支60 min建立MI模型。以超声确定MI后两周,采用心肌注射器经心内膜向心肌内注入1×108个小型猪自体红色荧光染料Dil标记的BMSCs,并观察心电图(ECG)的变化。移植BMSCs 8周后,取心肌组织行病理切片,选取含有移植细胞的切片行免疫荧光染色。结果: 4只小型猪成功地建立MI模型（1只因术中室颤死亡）并完成经心内膜的细胞移植,注射过程可出现一过性室性早搏以及短阵室速。通过心肌免疫荧光染色证实,移植的BMSCs已在宿主心肌内存活。结论: 使用心肌注射器经心内膜心肌移植自体BMSCs创伤小,方法可行,具有临床应用的前景。     

Differences in time course of myocardial mRNA expression in non-infarcted myocardium after myocardial infarction

In non-infarcted myocardium after myocardial infarction, the change of cardiac phenotypic modulation of contractile protein, extracellular matrix and intracellular Ca²⁺ transport protein, such as sarcoplasmic reticulum Ca²⁺(SR-Ca²⁺)-ATPase, Na⁺-Ca²⁺ exchanger, have a important role during cardiac remodeling. However, the time course in this gene expression in the adjacent and remote left ventricular, or right ventricular myocardium after myocardial infarction has not been well examined. The purpose of this study was to examine the left ventricular function and regional cardiac gene expression after myocardial infarction. Myocardial infarction was produced in Wistar rats by the ligation of the left anterior descending coronary artery. After 3 weeks, 2 months and 4 months from myocardial infarction, we performed Doppler echocardiography and measured the systolic and diastolic function. Then, we analyzed the contractile protein, extracellular matrix and intracellular Ca ²⁺ transport protein mRNAs of cardiac tissues in the adjacent and the remote noninfarcted myocardium, and right ventricular myocardium by Northern blot hybridization. Fractional shortening of infarcted heart progressively decreased. Peak early diastolic filling wave (E wave) velocity increased, and the deceleration rate of the E wave velocity was more rapid in myocardial infarction areas. Atrial filling wave (A wave) velocity decreased, resulting in a marked increase in the ration of E wave to A wave velocity. Expression of myocardial α-skeletal actin, β-MHC and ANP mRNA, or collagen I and III mRNA were higher at 3 weeks after myocardial infarction. SR Ca²⁺-ATPase mRNA in the adjacent non-infarcted myocardium was decreased at 2 months, and that in remote myocardium was decreased at 4 months after infarction. Na⁺-Ca²⁺ exchanger mRNA levels were increased at 3 weeks, but was decreased at 2 months in the adjacent non-infarcted myocardium and at 4 months in the remote myocardium. These findings suggest that the compensation for myocardial infarction by myocardial gene expression in non-infarcted myocardium may occur at an early phase after myocardial infarction, and myocardial dysfunction may begin from adjacent to remote non-infarcted myocardium during progressive cardiac remodeling. Received: 9 August 1999, Returned for revision: 16 September 1999, Revision received: 5 January 2000, Accepted: 26 January 2000     

多普勒组织成像检测冠心病患者梗死心肌的研究

目的　测算比较正常室壁心肌和梗死心肌的运动速度 ,评估多普勒组织成像 (DTI)对冠心病梗死心肌的诊断价值。方法　心肌梗死患者 4 6例 ,分成前壁梗死组 2 9例和下壁梗死组 17例 ,健康人 4 8例作对照 ;在心尖左室长轴切面 (alax)上测得室壁节段厚度 ;于心尖四腔心切面(ap4cv)、心尖二腔心切面 (ap2cv)、胸骨旁左室长轴切面(pslax)和alax应用DTI ,按左室壁 16节段 ,测量各室壁节段心肌运动曲线的速度指标 :收缩期峰值运动速度 (Sm) ;舒张早期峰值运动速度 (Em) :收缩期峰值速度梯度 (PVGs) :舒张早期峰值速度梯度 (PVGe) :收缩期跨壁速度梯度 (MVGs) :舒张早期跨壁速度梯度 (MVGe) ,并分别作组间和组内比较。结果　 (1)心梗患者内膜下心肌绝大多数节段性的室壁运动速度降低 ,与对照组存在显著差异 (P <0 0 1或 <0 0 5 ) :(2 )前壁梗死患者前间隔峰值速度梯度 (PVG)与跨壁速度梯度、(MVG)降低 ,中间段明显降低 (P <0 0 1或 <0 0 5 ) ,后壁基底段的MVGs 显著升高 (P <0 0 5 ) ;前间隔PVG、MVG明显小于后壁 (P <0 0或 <0 0 5 ) ;(3)下壁梗死患者各室壁节段舒张早期峰值速度梯度 (PVGe)除后壁基底段外均降低 ;同水平节段内膜下心肌峰值运动速度 (Sm、Em)趋于一致。结论　梗死心肌运动速度明显低于正常     

Comparative study of stromal cell lines derived from embryonic,fetal, and postnatal mouse blood-forming tissues

OBJECTIVE: To better understand the differentiation of stromal cells of the hematopoietic microenvironment, we set out to characterize stromal cells from the different developmental sites of hematopoiesis in the mouse (30 bone marrow, 7 spleen, 3 embryonic and 15 fetal liver, 6 yolk sac, and 6 aorta-gonad-mesonephros lines) for expression of 22 cytoskeletal, membrane, and extracellular matrix proteins. MATERIALS AND METHODS: Western blotting, immunofluorescence, and flow cytometry were used. Statistical methods included principal components analysis and analysis of variance. RESULTS: Stromal cells from 11 dpc mouse embryos express mesenchymal and vascular smooth muscle cell (VSMC) markers. Principal components analysis on the 70 stromal cell lines isolated from different anatomic sites and developmental stages allows classification of stromal lines along a mesenchymal to VSMC differentiation pathway. Stromal cells do not express endothelial and hematopoietic differentiation membrane antigens, but they do express integrin alpha(5), alpha(6), and beta(1) subunits, vascular cell adhesion molecule-1, CD44, stem cell antigen-1, Thy-1, CD34, and endoglin. The intensity of expression of certain markers differs between lines according to the anatomic site of origin. CONCLUSIONS: This study indicates that stromal cells, whatever their anatomic site of origin, follow a VSMC differentiation pathway, suggesting a blood-forming tissue-specific differentiation of mesenchymal stem cells. Differential quantitative expression of distinct sets of markers appears to be correlated with the anatomic sites of origin of the stromal cells.