Rudolf Vracko

Skeletal Muscle Capillaries in Diabetics A Quantitative Analysis

Capillary sizes, the amounts of endothelium, pericytes, basal lamina (BL), and lumina were measured with planimeter on electron micrographs of 20 capillary cross sections from each of 40 skeletal muscle specimens obtained from any of eight specific body sites of 20 diabetics. While the capillaries in the distal abdomen and the proximal thigh do not have thickened BL, those in the muscles of mid-abdomen, chest wall, hand, mid-thigh, mid-lower leg, and foot have significantly increased amounts of BL. The remaining parameters differ among body sites, generally following a pattern established earlier for the skeletal muscle capillaries of nondiabetics. Because of the focal and regional distribution of BL thickening and because it occurs also in nondiabetics, it is concluded that BL thickening is a common but not characteristic feature of diabetes mellitus. A possible mechanism which may lead to BL thickening is proposed and discussed.

Nerve fibers in human myocardial scars

The relationships between ischemic heart disease, myocardial scars, ventricular nerve fibers, and ventricular arrhythmias have not been established despite considerable evidence suggesting important correlations. We recently described the reactions of nerve fibers in necrotic, healing, and healed rat myocardium. Prompted by these studies and by the lack of similar information for humans, we studied the structural relationships between nerve fibers and human myocardial scars. Hearts were obtained from transplant surgery and autopsy. Nerve fibers were labeled with antibody to S-100 protein. Light and electron microscopy of left ventricular scars revealed (1) fiber densities greater than those in adjacent intact myocardium, (2) fiber aggregates concentrated irregularly along the periphery of lesions, (3) fibers few in number or absent in the deeper aspects of scars, and (4) axonal enlargements containing clear and dense storage granules within the fiber aggregates. Like all other elements of the scars, the nerve fibers appeared to be oriented predominantly in the long axis of myocytes located at the edges of the lesions. Based on our experimental findings in rat hearts, these studies suggest that human myocardial nerve fibers regenerate after necrotizing injuries and that at least some of the resulting scar-associated fibers have structural features differing from those in uninjured myocardium. We suspect that these structural differences might be associated with functional alterations that could affect the triggering of ventricular arrhythmias.

Mechanical thrombolysis: a new rotational catheter approach for acute thrombi.

We tested a new rotational thrombectomy catheter in acute thrombi formed both in vitro and in vivo. The catheter consisted of a rounded platinum tip, 0.025 inch diameter by 0.08 inch long, attached to a flexible steel guidewire supported by an external sheath. In vitro, the force required to penetrate thrombus was reduced fivefold by rotation of the catheter at 4000 rpm (0.75 +/- 1.2 g rotating vs 3.9 +/- 2.1 g static; p less than .001). Fibrin was extracted selectively from the thrombus and tightly wound about the shaft (3.8 +/- 1.5 mg rotating vs 0.75 +/- 0.4 mg static; p less than .001). In vivo, subtotal or complete thrombosis of the canine femoral artery was created. Thrombectomy by catheter rotation always produced tightly wound adherent fibrin on the catheter shaft. Angiographic patency was restored in 20 of 22 (91%) arteries, totally in seven of 22 (32%) and partially (greater than 20% increase in lumen diameter) in 13 of 22 (59%). There was one arterial perforation (5%). We conclude that this new mechanical catheter device reduces the force required to penetrate thrombus. Additionally, by winding fibrin about its shaft, the catheter is able to selectively remove the fibrin matrix of thrombus. Thus both the ease of initial thrombus recanalization as well as physical removal of thrombus are promoted by this new approach. Such an approach may be relevant to the treatment of recent thrombosis in acute myocardial infarction.

Skeletal Muscle Capillaries in Nondiabetics

To evaluate the variations of capillary calibers and those of basal lamina (BL), pericytes, endothelium, and lumen within a sample of skeletal muscle and among samples from different anatomic sites, areas occupied by total capillary cross sections, by the lumen, endothelium, pericytes, and BL were measured on electron micrographs. Twenty capillary cross sections from each of the 41 specimens of skeletal muscle from 23 nondiabetic males were used.For all parameters, a marked variation was noted within each specimen, between specimens from identical sites of different individuals, and between different anatomic sites of the same individuals. In general, the capillaries are largest in the lower leg and foot muscles; the lumen is smallest at the levels of mid-thigh and lower leg; the capillaries of the lower extremity have a greater investment of BL, endothelium, and pericytes than the capillaries of the trunk and hand muscles. The areas occupied by endothelial cytoplasm, pericyte cytoplasm, and by BL are larger in bigger capillaries apparently owing to greater circumference. In addition, the capillaries of the leg, particularly those of the lower leg, have an absolutely greater investment of BL than capillaries from proximal sites. Reasons for these regional differences in the overall size of capillaries and in the investment of BL are not known.

Overview Article: Basal Lamina of Epidermis, Muscle Fibers, Muscle Capillaries, and Renal Tubules: Changes with Aging and in Diabetes Mellitus

Using autopsy material from 14 diabetics and 10 controls (8-84 years old), the authors measured the thickness of basal lamina (BL) in skeletal muscle capillaries, renal tubules, skeletal muscle fibers, and epidermis to determine whether BL accumulation is a generalized phenomenon or limited only to certain anatomic structures. The four structures were selected because earlier experiments in animals have shown that in two (muscle capillaries and renal tubules) BL accumulates as a by-product of cell renewal while in the other two (muscle fibers and epidermis) it does not. In human tissues we found that BL accumulates in muscle capillaries and renal tubules but not in muscle fibers and epidermis, in muscle capillaries and in renal tubules it accumulates in controls and in diabetics as a function of aging, more BL in both anatomic structures accumulates in diabetics than in controls, and the extent of BL accumulation in muscle capillaries and renal tubules does not correlate with duration of diabetes mellitus. In addition to the fact that BL does not accumulate in all anatomic structures in which BL is normally present, the observations indicate that diabetes alone is not responsible for BL accumulation, diabetes exaggerates age-dependent accumulation of BL, and accumulation of BL in man is probably a by-product of cell renewal.

Spatial arrangements of microfibrils in myocardial scars: Application of antibody to fibrillin

Acute myocardial infarction kills myocytes; viable and necrotic myocytes disconnect and the ends of the viable cells become anchored to collagen fibers during reparative scar tissue formation. These anchorages have not been examined in detail, although previous studies have shown that microfibrils (MFs) concentrate at the edges of scars and at the tips of normal papillary muscles. We examined the spatial arrangements of MFs at these two sites in human hearts. Light and electron microscopic observations were made on tissue samples oriented in the long axis of myocytes and stained with monoclonal antibodies to fibrillin, a glycoprotein component of human microfibrils. MFs had identical arrangements at both sites, where they formed fibrous connections between myofibers and collagen fibers. These connections were oriented in the long axis of the muscle cells. At the myocyte ends, MFs appeared to intertwine with MFs in the normal endomysium; in the main body of the connections, MFs formed compact, 200 to 500 nm thick, fibrillin-positive fibers; and at the collagen ends, MFs splayed out among collagen fibrils. These observations indicate that MFs form myofiber-collagen fiber linkages at sites where the power of myocardial contraction is being transmitted to the extracellular connective tissue framework. Formation of such linkages seems to be an important step in the successful repair of necrotic myocardial lesions.

Lifespans of diabetic and non-diabetic fibroblasts in vitro: Results of replicate determinations☆

Abstract Cumulative replicative lifespan (CRLS) was determined in replicates for skin fibroblasts from ten non-diabetic and 11 diabetic donors. The results show that CRLS 1. 1. decreases as function of donor age for controls but not for diabetics 2. 2. is not different for random subsites of the same biopsy 3. 3. is not affected by freeze-thawing of either tissues or cells or by the calendar time during which the cells were tested 4. 4. is affected by different lots of fetal bovine sera. The data also shows that variation of CRLS among diabetic donors is greater than among controls and that cells from some diabetics may have decreased CRLS.

Myofibroblasts and smooth muscle cells in human myocardial scars: Possible origins and inductive factors

Necrotic myocardial lesions, including myocardial infarcts, heal by formation of scar tissue, and it is commonly thought that fibroblasts are the principal cells producing and maintaining the scar tissue matrix. In contrast to this notion, our study of old myocardial scars in 11 human hearts revealed that myofibroblasts (MFBs) and nonvascular smooth muscle cells (SMCs) were the dominant cell types. Both contractile cell types stained positively with antibody to α-smooth muscle actin and were identified ultrastructurally by the presence of nonsarcomeric myofibrillar elements. They were distinguished from each other by basal lamina, which invested the SMCs but not the MFBs. Neither cell type was identified in the myocardium of 3 normal human hearts, where fibroblasts were the normal interstitial cells. In earlier studies, we made similar observations in rat heart scars. The presence of MFBs and SMCs in myocardial scar tissue but not in normal myocardium raises questions about their origins, lineage relationships, inductive mechanisms, and functions. The results of our studies indicated that the MFB and SMC phenotypes were induced and maintained by the cyclical physical forces associated with myocardial contractions; that fibroblasts and MFBs seemed to be phenotypic modulations of the same cell type in a physical environment favoring expression of the MFB form; that the nonvascular SMCs were not derived from MFBs but may have come from cells located in vessel walls; and that the contractile cells in scar tissue might resist and counteract the stretching forces induced by myocardial contractions.

Combined mechanical and chemical thrombolysis in an experimental animal model: Evaluation by angiography and angioscopy

In an experimental animal model of femoral artery thrombosis, contrast angiography was compared to intravascular angioscopy. Additionally, the effect of mechanical, rotational thrombectomy and the additive benefit of the administration of intravascular streptokinase were assessed by means of both procedures. After external forceps crush injury alone, contrast angiograms were generally normal (6 of 14) or showed minimal luminal irregularity (3 of 14), and 5 of 14 had 30% to 50% stenosis. With angioscopy, none appeared normal, and 14 of 14 showed thrombi layered along the wall, as well as intimal flaps, and 6 of 14 had partially occlusive thrombi (p less than 0.001 angiography vs angioscopy). After 2-hour occlusion and injection of thrombin into the injured segment, angiographic total (5 of 14), subtotal (3 of 14), or partial thrombotic occlusions (5 of 14) were created. Angioscopy showed similar results, except that total occlusions were classed as subtotal occlusions. After rotational thrombectomy, most arteries again appeared normal by contrast angiography (6 of 11) but none were angioscopically normal (p less than 0.006). Streptokinase, administered after rotational thrombectomy in seven arteries, normalized one 30% angiographic stenosis; there were no other angiographic changes. Findings with angioscopy were also unchanged. We conclude that in the diagnosis and treatment of intravascular thrombosis, angioscopy is generally more sensitive in the detection of intravascular thrombi, with the exception of total thrombotic occlusions. Angioscopy was uniquely effective in identifying subintimal flaps, which were never identified by angiography. In this model, streptokinase provided little or no additional thrombolytic benefit to mechanical thrombectomy alone.

SEEDING EFFICIENCY, PLATING EFFICIENCY, AND POPULATION DOUBLINGS OF HUMAN SKIN FIBROBLASTLIKE CELLS: RESULTS OF REPLICATE TESTING

SummaryUsing skin fibroblastlike cells from 10 healthy volunteers, 12 to 54 yr old, we measured, in replicates, seeding efficiency (SE), plating efficiency (PE), and population doublings (PD) during 2 wk of growth and the cumulative replicative life span (CRLS). We determined the effects of donor age and of in vitro cell age on the test results and test precision. We found that (a) On the average, SE was 82%±24 SD, PE was 12%±9 SD, PD was 7.3 doublings ±2.5 SD, and CRLS was 31.1 doublings ±9.6 SD. (b) Plating efficiency and PD correlated well with each other and with CRLS whereas SE did not correlate with PE, PD, or CRLS. (c) Plating efficiency and PD were affected negatively by increasing passage levels and by donor ages, but the influence of age was evident only for cells from early passages. Seeding efficiency was not affected by either of the two variables. (d) Variations in PD from early passages explained half of the donor-to-donor variation in CRLS whereas PD from later passages had little or no predictive power. (e) Of the two short term tests, PD had better precision than PE. For replicate runs with cells from early passages the coefficient of variation was 44% for PE and 15% for PD.The results show that PD for cells from early passages in mass culture give rapid results, which express the in vitro replicating potential of cells from different donors with greater precision than do measurements of CRLS.