Biomedical subjects

R Vracko

Publications and source records attributed to R Vracko.

At least 19 recordsLinked to original sources

PubMed1994

Macrophages express osteopontin during repair of myocardial necrosis.

Osteopontin is a secreted glycoprotein implicated in a variety of functions, including cell adhesion and migration. Because these functions may be of general importance in the response of tissue to injury, we examined osteopontin expression after experimental cardiac injury and human myocardial infarction. Rat hearts were injured by transdiaphragmatic freeze-thaw and examined from 1 to 28 days after injury. Osteopontin was absent from normal myocardium by immunocytochemistry, Western blotting, and in situ hybridization. On days 1 and 2 after injury, osteopontin mRNA and protein were expressed at high levels by macrophages infiltrating necrotic myocardium. Double labeling with the macrophage marker ED1, however, demonstrated that only a subset of macrophages expressed osteopontin. Western blot analysis showed a single 66-kd band in injured myocardium that was absent from control tissue. Although macrophages remained abundant in the ensuing granulation response and scar tissue formation, the expression of osteopontin was diminished on day 4 and markedly downregulated at 1 and 4 weeks after injury, with only rare cells expressing the message or protein. In a human heart with an 8-day-old myocardial infarct, there was abundant expression of osteopontin mRNA and protein in macrophages within the necrotic and granulation tissue. Transient expression of osteopontin was also observed in a subset of macrophages infiltrating lung, skin, and skeletal muscle injured during the experiment, indicating the response was not limited to the heart. Thus, synthesis of osteopontin by macrophages appears to be a generalized response in the reaction to tissue injury. Although macrophages persist in these lesions, osteopontin is dramatically downregulated as healing proceeds. These results provide the first evidence that osteopontin may be important in healing after tissue injury, possibly in cellular adhesion, chemotaxis, and/or phagocytosis.

Animals↗

PubMed1992

Noninvasive arterial thrombus imaging with 99mTc monoclonal antifibrin antibody.

BACKGROUND: The T2G1s monoclonal antifibrin antibody binds specifically to fibrin but not to fibrinogen. METHODS AND RESULTS: In a canine model of acute arterial thrombosis, we determined the feasibility of imaging thrombi using a 99mTc-labeled Fab' fragment. In 14 dogs, 10 carotid and 13 femoral artery thrombi were produced using 2-hour temporary occlusion, crush injury, and local thrombin injection methods. A sham-operated carotid artery served as control. Antifibrin antibody was injected intravenously at the end of temporary occlusion. Serial planar radionuclide images were obtained immediately and at 1 and 2 hours. Following killing the dogs at 2 hours, we measured antibody uptake ex vivo in 5-mm-long segments of thrombus, the adjacent injured artery, and a control artery. Antibody was cleared from the blood with a mean +/- SD t1/2 of 121 +/- 23 minutes. The thrombi weighed 218 +/- 140 mg. Antibody uptake in the thrombi was patchy, and the thrombi were closely adherent to the injured arterial wall. In the segment with maximal ex vivo antibody uptake, the ratio of control artery to blood counts/g/sec was 0.65 +/- 0.46, the injured artery-to-blood ratio was 2.35 +/- 1.01 (p less than 0.0001 versus control), and the thrombus-to-blood ratio was 4.24 +/- 2.58 (p less than 0.0001 versus control). In three dogs, an isotype-matched ovarian tumor antibody labeled with 111In was injected with T2G1s but was not taken up in the thrombus or the adjacent arterial wall. Visual analysis of the in vivo carotid radionuclide images showed uptake by 2 hours in all 10 carotid thrombi. Quantitative image analysis, measured as the thrombus-to-opposite carotid artery ratio, showed increasing uptake over time with ratios of 1.1 +/- 0.3, 1.6 +/- 2.0, and 2.2 +/- 1.3 on the immediate, 1-hour, and 2-hour images, respectively. All quantitative ratios of 1.3 or greater were visually identified. CONCLUSIONS: 99mTc-labeled Fab' fragments of the T2G1s antibody are taken up specifically by acute arterial thrombi after intravenous injection. Uptake is progressive over a 2-hour period, and all thrombi are detected by radionuclide imaging at 2 hours. These results show that it is feasible to noninvasively detect arterial thrombi within 2 hours of formation.

Animals↗

PubMed1991

Thrombolysis by rotational thrombectomy followed by tissue plasminogen activator: evaluation by angioscopy.

Thrombus removal using percutaneous rotational thrombectomy (PRT), followed by tissue plasminogen activator (t-PA), was studied by contrast angiography and fiberoptic angioscopy in a canine femoral artery model of thrombosis. After thrombus induction and following each treatment, comparisons were made between angioscopy and angiography for the detection of thrombus and subintimal dissection. Angioscopic images were scored in a blinded fashion for lining, protruding, or occlusive thrombus (class 1,2, or 3) as well as estimated wall coverage by thrombus. Angiograms were studied for percent diameter stenosis and the presence of flaps. Following external forceps crush injury of 18 arteries, two hour occlusion, and injection of thrombin, mean angiographic stenosis was 66%, thrombus coverage by angioscopy was 81%, and mean angioscopy class was 2.5. Following PRT, stenosis decreased to 27% (p less than 0.008), thrombus coverage was reduced to 49% (p less than 0.02), and angioscopy class dropped to 2.0 (p less than 0.07). After t-PA treatment, these values were further reduced to 25% (p = NS), 26% (p less than 0.02), and 1.3 (p less than 0.008), respectively. In comparison to angiography, subintimal dissection (seen as flaps) and thrombus (lining, protruding, or occlusive) were present significantly more often by angioscopy (p less than 0.001). It is concluded that PRT results in significant thrombolysis, apparent by angiography and angioscopy. Follow-up t-PA can produce additional, incremental thrombolysis, apparent only by angioscopy. A beneficial role for t-PA following mechanical thrombolysis is suggested by this model. The superior sensitivity of angioscopy for detection of flaps and thrombus is underscored by this study.

Animals↗

PubMed1991

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.

Adult↗

PubMed1991

Contractile cells in rat myocardial scar tissue.

In earlier studies of rat myocardial tissue reactions to necrotizing injuries, we observed in the resulting scar tissue a large number of smooth muscle cells unassociated with blood vessels. Since these cells are not normal components of ventricular myocardium, we studied the appearance and fate of all cells with smooth muscle-like features in healing and healed lesions at intervals up to 10 weeks after ischemic or freeze-thaw injuries. Observations were made with light and electron microscopes, using conventional methods and immunostaining methods to detect alpha-smooth muscle actin. In healing and healed lesions, smooth muscle actin was detected histologically in capillary pericytes, myofibroblasts, and both vascular and nonvascular smooth muscle cells. Its association with cytoplasmic microfilaments in nonsarcomeric myocytoskeletal arrangements was confirmed ultrastructurally. The pericytes and myofibroblasts predominated during the earlier hypercellular healing period. The smooth muscle cells appeared near the end of the first week of repair; they were initially located mainly in presumptive vascular structures, identified by residual basal lamina sheaths, but subsequently located mainly in nonvascular locations. After the second week until the end of the study the number of nonvascular smooth muscle cells increased and that of myofibroblasts decreased. The nonvascular smooth muscle cells predominated in the larger mature scars, especially the transmural ones. From these observations, we have concluded that contractile cells other than cardiac myocytes have important roles in myocardial tissue repair, have suggested that their roles are related to the forces of myocardial contractions, and have discussed their possible functions and lineage interrelationships.

Actins↗

PubMed1990

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.

Adult↗

PubMed1990

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.

Angiography↗

PubMed1990

Fate of nerve fibers in necrotic, healing, and healed rat myocardium.

Myocardial infarction, myocardial scar tissue formation, and cardiac arrhythmogenesis seem to be associated. There are electrophysiological data suggesting that myocardial nerves are involved in arrhythmia development; however, there are no morphologic studies describing the fate of these nerves following necrotizing myocardial injuries. To describe the reactions of Schwann cells and axons following such injuries, we induced lesions in rat hearts with ischemia or transdiaphragmatic freeze-thawing and examined the acutely necrotic, healing, and healed lesions with light and electron microscopy. Antibodies to Schwann cell-associated S-100 protein were used to facilitate histologic detection. Both forms of injury produced focal lesions in which Schwann cells were killed and axonal segments destroyed; however, the basal lamina sheaths of cardiac myocytes and capillaries, and probably also of nerve fibers, remained largely intact. During 4 weeks of sequential observations, Schwann cells and axons were components of a hypercellular healing front that began at the periphery and moved toward the center of each lesion. Their proliferation and growth may have occurred within the original nerve basal lamina sheaths, and reparative axonal enlargements contained an abundance of 50- to 100-nm clear and dense storage granules. Fully developed nerve fibers were not only present in newly formed scar tissue but also appeared to be present in significantly greater density than in uninjured myocardial tissue. These findings demonstrate that proliferative nerve fiber regeneration occurs from the edges of necrotizing myocardial injuries, that healing results in relatively large number of nerve fibers in newly formed scars, and that axons in these scar-associated nerve fibers contain an abundance of neurosecretory granules. The functional significance of these observations remains to be determined.

Animals↗

PubMed1989

Rotational thrombectomy in acute canine coronary thrombosis.

We have developed a mechanical thrombolytic catheter which defibrinates a fresh intra-arterial thrombus by wrapping fibrin about its rotating shaft. Defibrination results in liquification of the thrombus and reperfusion of the thrombotically occluded vessel. In this study, we employed this catheter-based approach in dogs with coronary thrombosis to simulate possible clinical use in acute myocardial infarction. Total coronary thrombosis was generated in 11 dogs. Spontaneous reperfusion did not occur over a 30-minute control period. All vessels studied were initially totally thrombosed. After mechanical thrombolysis, there was a significant improvement in percent diameter stenosis from 100% to 28 +/- 26% (P less than 0.001). After thrombolysis, angiographically graded blood flow was normal in 9 of 11 arteries and was mildly delayed in 2 of 11. Complications included perforation of 2 vessels. We conclude that mechanical thrombolysis, with a rotating catheter, results in prompt reperfusion of the infarct vessel and significant improvement in distal blood flow. This approach, unlike angioplasty, removes the thrombus and might serve as an alternative to or supplemental form of mechanical thrombolysis.

Angioplasty, Balloon↗

PubMed1989

Connective tissue cells in healing rat myocardium. A study of cell reactions in rhythmically contracting environment.

To better understand the tendency of myocardium to heal by scarring rather than regeneration, the authors examined the responses of connective tissue cells (CTCs) after three types of necrotizing injuries. Derived from myocardial interstitial cells, CTCs proliferated in both the connective tissue space and the compartment of necrotic myocytes. They assumed various cell forms: fibrocytelike CTCs throughout the sites of injury deposited extracellular scar tissue elements, established CTC-myocyte contacts, and helped anchor myocytes to scar tissue with myotendonlike specializations; CTCs with more complex forms established CTC-myocyte relationships, suggesting important roles in communication and tissue remodeling. CTCs within scar tissue differentiated into myofibrocytes, chondrocytes, and possibly smooth muscle cells. Most scar tissue elements were disposed in the long axis of myocytes. These alterations in form indicate that CTCs have various roles in myocardial repair and suggest that a number of the roles are modulated by contractile forces.

Animals↗

PubMed1988

Rotational atherectomy in atherosclerotic rabbit iliac arteries.

PTCA is not technically possible in many patients with symptoms of coronary artery disease. In addition, atheroma is not physically removed by PTCA, and restenosis of the treated vessel is common. We have tested a new, rotating, abrasive-tipped angioplasty device in vivo in 13 atherosclerotic rabbit iliac vessels. Atherosclerosis was generated in rabbit iliac vessels by a 2% cholesterol diet combined with balloon endothelial injury for 10 weeks. The diseased vessels were then treated with the rotational atherectomy device. Before treatment, contrast angiograms demonstrated that initial percent diameter stenosis was 81% +/- 9%. After atherectomy, there was significant improvement, with residual 38% +/- 22% narrowing (p less than 0.001). One perforation resulted from distal guidewire manipulation, and one vessel was occluded by the device. Histologic examination demonstrated loss of portions of the diseased intima in all cases. Particles were produced for analysis in vitro by operating the atherectomy device in atherosclerotic rabbit aortas perfused with saline solution. Ninety-eight percent of the particles produced by the device were less than 10 micron in diameter. We conclude that this new rotational device can remove atheromatous material from diseased arteries in rabbits. Such a device may complement other angioplasty techniques and lead to wider application of catheter-based therapeutic interventions.

Animals↗

PubMed1988

Basal lamina of rat myocardium. Its fate after death of cardiac myocytes.

As part of a study of the interactions between myocardial cells and extracellular matrix during healing of necrotic lesions, we have examined the fate of myocyte basal lamina (BL) after injury with ischemia, freeze-thawing, or isoproterenol. Using light and electron microscopy, and antibodies to three BL-associated antigens, we found that the BL of necrotic myocytes remained largely intact and continued to delineate the myocyte compartment from connective tissue space. Inflammatory cells entered the myocyte compartment through holes in the acellular BL and removed cell debris. The holes may have been produced by inflammatory cells and/or by the stretching force of the beating heart. After removal of debris, some BL sheaths of necrotic myocytes collapsed, resulting in spatial approximation of vessels. Interstitial cells deposited collagen and elastic fibers in the connective tissue space and within portions of the myocyte compartment. The acellular myocyte BL, collapsed or not, retained normal antigen staining for type IV collagen, laminin, and heparan sulfate for about 10 days, then showed diminished staining in patchy areas. These areas may correspond to BL disruption and degradation in conjunction with fibrosis, although a substantial amount of acellular BL remained in situ and became embedded in scar tissue. At least two types of granulo-vesicular bodies, measuring 25 to 60 and 60 to 160 nm respectively, were associated with the acellular BL; these were of unknown origin and function. The study shows that the fate of acellular BL in injured myocardium is similar to the fate of BL in other injured tissues; however, the appearance of holes in acellular BL, within hours after injury, is unusual and may enhance scar tissue formation. Whether the acellular BL contributes to regeneration of myocardium, as do acellular BLs in other injured tissues, remains to be determined.

Animals↗

PubMed1988

Myocyte reactions at the borders of injured and healing rat myocardium.

To better understand the apparent tendency of myocardium to heal by scarring rather than by restoration of normal structure, we have examined by light and electron microscopy the basic reactions of rat ventricular myocytes and their interactions with the extracellular matrix. Using three different types of necrotizing injuries, we found that necrotic myocytes separated from viable myocytes at intercalated discs leaving blunt-ended stumps at the edge of each lesion; the basal lamina of necrotic myocytes remained largely intact and spanned the gap between viable myocytes on opposite sides of each lesion. A small number of stumps were capped off by a new layer of basal lamina and showed no evidence of proliferative activity. The majority of the stumps developed cell processes that extended along the acellular myocyte basal lamina sheaths. These processes had one of two different fates. Some became apposed to similar processes, formed intercalated disc attachments, increased myofibrillar mass, and appeared to be associated with muscle reconstruction. Others developed elongate tapered ends, which terminated in myotendinous connections to scar tissue. The outcome of healing necrotic myocardium, like the healing of noncardiac necrotic tissue injuries, appears to be a function of cell growth and extracellular framework guidance; however, unlike healing of noncardiac tissues, healing of myocardium is uniquely complicated by continuing muscle contractions. We think the rhythmic pulling at the edges of necrotic lesions induces formation of myotendinous attachments, which anchor myocytes to scar tissue and probably prevent further growth.

Animals↗

PubMed1987

Mechanical thrombectomy: a comparison of two rotational devices and balloon angioplasty in subacute canine femoral thrombosis.

In this study, two prototype rotational devices were compared to balloon angioplasty in a canine model of subacute arterial thrombosis. Radiographic 2- to 8-day-old total thrombotic occlusions were produced in 30 canine femoral arteries. A high-speed rotating device with a cutting tip was used in 18 arteries. Successful opening occurred in every case, with a residual percent diameter stenosis at 45 +/- 25%. Vessel perforation was seen in 6 of the 18 arteries. A noncutting rotational thrombectomy catheter was used in six arteries. Radiographic patency was established in two of six (residual stenosis 86 +/- 28%), with one perforation with the use of the noncutting thrombectomy catheter. Balloon angioplasty reestablished radiographic patency in three of six arteries (residual stenosis 77 +/- 2%). No perforations were seen with balloon dilation, but radiographic distal emboli were always observed. No radiographic emboli were observed with either of the rotational devices. We conclude that subacute arterial thromboses are easily opened with an abrasive-tipped rotating angioplasty device. Although perforations are relatively common with this prototype equipment, design changes may produce a clinically useful angioplasty device.

Angioplasty, Balloon↗

PubMed1986

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.

Acute Disease↗

PubMed1985

Freeze-thaw injury of rat heart across an intact diaphragm: a new model for the study of the response of myocardium to injury.

A new method for producing a circumscribed injury in rat myocardium is described. It utilises laparotomy and freeze-thawing across an intact diaphragm. Morbidity and mortality are negligible because the chest and pericardial cavities, the major coronary vessels, and the major branches of the conduction system remain intact. The size and location of the injury are reproducible and its sharp delineation facilitates accurate collection of samples from areas of interest. The myocytes are killed in the area of injury, whereas the capillary, nerve and connective tissue cells are killed centrally but preserved peripherally in a perimeter zone of injury in which capillaries remain patent. The reparative activities are initiated at the border between injured and uninjured myocardium; as they progress over the ensuing 6 weeks, the volume of damaged myocardium diminishes rapidly leaving only a rim of subepicardial scar tissue, and the thickness of ventricular muscle between the scar and endocardium returns almost completely to its pre-injury width. The mechanisms involved in the repair process are being investigated.

Animals↗

PubMed1984

Systemic absorption of oral cholestyramine.

A patient with Clostridium difficile -toxin colitis was treated with oral cholestyramine, but died of other causes 15 days later. At autopsy, the colitis had resolved, but cholestyramine particles were found within the vessels of most body tissues, most prominently in his ulcerated distal esophagus. Clusters of bacteria were found adjacent to some of the cholestyramine particles, suggesting a common portal of entry.

Administration, Oral↗

PubMed1983

Seeding efficiency, plating efficiency, and population doublings of human skin fibroblastlike cells: results of replicate testing.

Using 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.

Adolescent↗