Apoptosis during coronary artery orifice development in the chick embryo.

Previous studies regarding the development of proximal segments of the coronary arteries in the chick have demonstrated that these vessels do not develop as angiogenic outgrowths from the aorta. Rather, the proximal segments of the coronary arteries arise from a peritruncal capillary plexus in the epicardium that coalesces around the aortic and pulmonary outflow tracts. Vessels from the peritruncal plexus grow toward and attach to the aorta at about Hamburger and Hamilton (HH) Stage 32 to establish the definitive coronary circulation. Currently, little is known about the process by which patent connections are established between these peritruncal vessels and the aorta. The hypothesis that apoptosis is involved in the formation of the coronary artery orifices was tested in the present study. Aortic and periaortic tissue from HH 29-35 chick embryos was examined using routine light and electron microscopy and TUNEL assays. Apoptotic cells were observed in close spatial and temporal association with the invasion of peritruncal vessels into the aorta (HH 29-31), the initial formation of coronary orifices (HH 32-33), and the further development of the definitive coronary arteries and orifices (HH 34-35). Whereas the origin of these apoptotic cells and the specific factors regulating their death remain unknown, the results of the present study strongly correlate apoptosis with the formation of proximal coronary arteries and their orifices. Our findings suggest avenues for further research and indicate that factors involved in regulating apoptosis should be included in future models of coronary artery development.

Apoptosis of endothelial cells in vessels affected by cerebral vasospasm.

BACKGROUND: Cerebral vasospasm after subarachnoid hemorrhage is a prolonged contraction that leads to cerebral ischemia or infarction. Morphological studies of cerebral arteries during vasospasm have shown extensive necrosis of smooth-muscle cells and desquamation and dystrophy of endothelial cells. The mechanism of cellular death is unknown. METHODS: We report an observation of apoptotic changes in the cerebral arteries of a patient who died after suffering severe cerebral vasospasm caused by aneurysmal rupture. Subarachnoid hemorrhage and cerebral vasospasm were confirmed by computed tomography scanning and angiogram. Histological and immunohistological examinations for apoptosis were performed in cerebral arteries. For control, the arteries from another patient, who died of trauma without head injury, were used. RESULTS: Corrugation of the internal elastic lamina and increased amounts of connective tissue was demonstrated by light microscopy. Apoptotic changes, characterized by condensation of chromatin of the nucleus and detachment from the basal membrane, were found on transmission electron microscopy in endothelial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling reaction revealed positive staining of the nuclei of the endothelial cells. CONCLUSIONS: This study demonstrates that apoptosis occurred in the cerebral arteries in a patient who died of cerebral vasospasm. The possible role of apoptosis in cerebral vasospasm is discussed.

Posttraumatic cerebral vasospasm: clinical and morphological presentations.

Head injury is one of the leading causes of morbidity and mortality in the young population. Many factors complicate head injury and worsen an outcome. One of these factors is posttraumatic cerebral vasospasm. We studied 75 patients admitted to the University of Mississippi Medical Center with head injury. Their ages ranged from 14 to 67 years (mean 30 years, SD 11.63). Eighty percent of the patients were men, and 20% were women. Of these patients, 53 (70.6%) suffered severe blunt trauma, and 4 patients suffered gunshot wounds to the head. Four patients had mild head injury, and 14 had moderate head injury. Posttraumatic vasospasm was detected in 24 (32%) patients. Among these patients, 19 (79.2%) had severe closed head injury, 3 patients had moderate head injury, and 2 suffered gunshot wounds. The severity of the patient's respective condition was correlated with the development of posttraumatic cerebral vasospasm: 50% of the patients with Glasgow Coma Scale (GCS) 3-4 developed PTV, and only 30% with GCS 9-11, and none of the patients with GCS > 12 developed PTV. Overall, posttraumatic vasospasm started earlier and had a shorter course than did aneurysmal vasospasm. Morphologically, posttraumatic vasospasm resembled the features of aneurysmal vasospasm. We found increased corrugation of the internal elastic lamina and increased amounts of connective tissue in the subendothelial layer. These findings show that posttraumatic vasospasm, although clinically more mild, demonstrates the same morphological changes as aneurysmal vasospasm.

Oxyhemoglobin-induced apoptosis in cultured endothelial cells.

OBJECT: Oxyhemoglobin (OxyHb) is one of the most important spasmogens for cerebral vasospasm that follows aneurysmal subarachnoid hemorrhage. The cytotoxic effect of OxyHb has been documented in endothelial and smooth-muscle cells; however, the pattern of cell death--necrosis or apoptosis--as the final stage of cell damage has not been demonstrated. This study was undertaken to determine if OxyHb induces apoptotic changes in cultured bovine aortic endothelial cells. METHODS: Confluent bovine aortic endothelial cells were treated with OxyHb in a concentration- and time-dependent manner. Cell density was assayed by counting the number of cells that attached to culture dishes after exposure to OxyHb. To identify apoptotic changes, the investigators used three specific methods: DNA fragmentation (electrophoreses), the apoptotic body (transmission electron microscopy), and cleavage of poly (adenosine diphosphate ribose) polymerase (PARP [Western blotting]). CONCLUSIONS: Oxyhemoglobin decreased cell density in a concentration- and time-dependent manner. Analysis of DNA showed a pattern of internucleosomal cleavage characteristic of apoptosis (DNA ladder). Transmission electron microscopy demonstrated condensation of nuclei and apoptotic bodies in OxyHb-treated endothelial cells. Western blotting with the PARP antibody revealed that the 116-kD PARP was cleaved to the 85-kD apoptosis-related fragment. These results for the first time demonstrated that the OxyHb induces apoptosis in cultured endothelial cells.

Morphological changes after percutaneous transluminal angioplasty.

BACKGROUND: Percutaneous transluminal angioplasty (PTA) dilates constricted arteries at the circle of Willis to reverse cerebral ischemia caused by cerebral vasospasm. Although 90% of the patients show angiographic improvement after PTA, only 70% show clinical improvement. Why some patients do not improve after PTA is unknown. We report on a 48-year-old woman who failed to improve after PTA and died from aneurysm rerupture. Pathologic studies were performed to determine why PTA failed to reverse the symptoms of cerebral ischemia. METHODS: The arteries of the brain were studied by light microscopy using Gomori's trichrome stain. The arteries were also studied by scanning and transmission electron microscopy. RESULTS: The arteries that were dilated with PTA showed compression of the connective tissue, stretching of the internal elastic lamina, and a combination of compression and stretching of the smooth muscle. The small arteries and arterioles that had been treated with an infusion of intraarterial papaverine were constricted with a thickened intimal layer. CONCLUSION: The persistence of cerebral vasospasm in small and perforating arteries may contribute to the failure of cerebral ischemia to reverse after PTA.

Protein kinase C has two different major roles in lattice compaction enhanced by cerebrospinal fluid from patients with subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Compaction of extracellular matrix (ECM) lattices by cultured fibroblasts was accelerated by cerebrospinal fluid (CSF) from patients with subarachnoid hemorrhage (SAH). The rate of acceleration was significantly related to the clinical grade of vasospasm. However, the mechanism remains unclear. Evidence exists for an important role in cerebral vasospasm for protein kinase C (PKC). The purpose of this study was to help clarify whether PKC has a role in contraction of the ECM. METHODS: We studied the effects of a myristoylated PKC peptide inhibitor (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) (PKC peptide inhibitor), (5-isoquinolinesulfonyl)-homopiperazine (HA-1077) (inhibitor of protein kinase A, myosin light-chain kinase, and protein kinase G), 7-deacetyl-6-(N-ace-tylglycyl)-forskolin (forskolin) (adenyl cyclase activator), and diacylglycerol-lactone (DAG-lactone) (PKC activator) on fibroblast-populated collagen lattice compaction with or without CSF from SAH patients. Four sets of fibroblasts were used: three explanted from skin and one from cerebral artery. RESULTS: Moderate and high concentrations of PKC peptide inhibitor inhibited lattice compaction with or without acceleration by CSF. Low concentration of PKC peptide inhibitor enhanced acceleration by CSF but had no effects without CSF. HA-1077 could not inhibit lattice compaction. Forskolin inhibited compaction. DAG-lactone accelerated compaction in early phases. CONCLUSIONS: In the mechanism of acceleration of contraction of ECM under the influence of CSF, PKC seems to have two different roles. Protein kinase A and myosin light-chain kinase apparently play more minor roles than PKC in the mechanism, but no evidence was found of a role for protein kinase G activation in matrix compaction.

Nonmuscle arterial constriction after subarachnoid hemorrhage: role of growth factors derived from platelets.

Recent studies have shown that myofibroblasts cultured from spastic arteries after subarachnoid hemorrhage (SAH) can produce contractile forces and that cerebrospinal fluid obtained from SAH patients accelerates this contraction. Myofibroblast-populated collagen lattices were used to evaluate the roles of polypeptide growth factors considered to be released from intraluminal accumulated platelets. The myofibroblasts, obtained at autopsy from human cerebral arteries of victims of vasospasm, caused contractile forces by compacting collagen fibrils in the matrix. Transforming growth factor beta 1 (TGF-beta 1) and platelet-derived growth factors aa and bb (PDGFaa and PDGFbb) accelerated this contraction in a dose-dependent manner. In the presence of TGF-beta 1 at 1 or 10 ng/ml, PDGFaa at 10 ng/ml, or PDGFbb at 10 ng/ml, lattice areas were reduced to 69.5 +/- 1.9% (mean +/- standard deviation) (P < 0.001), 63.3 +/- 0.4% (P < 0.001), 66.5 +/- 3.0% (P < 0.01), or 74.4 +/- 1.7% (P < 0.01) of the control on Day 6, respectively. The combination of subthreshold doses of TGF-beta 1 and PDGFaa created a stimulatory effect that appeared to act synergistically. Furthermore, myofibroblast-populated collagen lattices made with cells preincubated with TGF-beta 1 showed more rapid compaction with or without the presence of stimulants such as post-SAH cerebrospinal fluid. These results suggest that, in addition to other possible factors in the post-SAH cerebrospinal fluid, growth factors derived from accumulated platelets may play an important role in arterial constriction caused by nonmuscle components after SAH, by single or multiple mechanisms.

Accelerated nonmuscle contraction after subarachnoid hemorrhage: culture and characterization of myofibroblasts from human cerebral arteries in vasospasm.

Cell culture lines from human cerebral arteries showing vasospasm after subarachnoid hemorrhage were established from three autopsy cases. Each culture line showed the ultrastructural characteristics of myofibroblasts. Decreased alpha-actin antigenicity, demonstrated using the anti-smooth muscle cell alpha-actin antibody, was observed in cultured cells possessing abundant F-actin. When incorporated into the three-dimensional collagen matrix in vitro, the cultured cells compacted the collagen lattice at a rate equivalent to that of human dermal fibroblasts. Lattice compaction was significantly accelerated by cerebrospinal fluid taken from patients with symptomatic vasospasm. Compaction was completely inhibited by the addition of 10(-6) mol/L verapamil or 100 U/mL heparin. Neither nimodipine (10(-5) mol/L) nor nicardipine (10(-5) mol/L) inhibited compaction, and endothelin (10(-6) mol/L) and potassium chloride (40 mmol/L) had no effect. The morphological change of cells in the collagen lattice suggests that both verapamil and heparin affect cellular motility, filopodial protrusion, and cell attachment. These data suggest that myofibroblasts in human cerebral arteries differ from medial smooth muscle cells and can generate a force rearranging the proliferated collagen matrix present after subarachnoid hemorrhage. This reorganization can contribute to, or be responsible for, sustained vasoconstriction. Consequently, current treatment for vasospasm may need to be reevaluated to include the nonmuscle components in the vessel wall.

Mechanism of action of balloon angioplasty in cerebral vasospasm.

Recent technical advances in interventional neuroradiology have made it possible to dilate cerebral arteries showing vasospasm after a subarachnoid hemorrhage. Although the reported effects of dilatation in clinical cases have been dramatic, few experimental studies of the mechanism of action have been performed. It also is still unclear why dilated arteries rarely show restenosis. Using the scanning electron microscope, we examined changes in the three-dimensional structure of connective tissues in vessel walls after balloon angioplasty. Femoral arteries from cats and middle cerebral arteries from human autopsies were studied. The vessels were dilated in situ with a balloon catheter until the intimal pressure reached 1.5 Wr 3 atm; then they were fixed and digested with 88% formic acid. The specimens were freeze dried and observed under the scanning electron microscope. Normal vessels without balloon dilatation were treated in the same manner and used as controls. The results showed that the normal structure of collagen fibers in the vessel walls was affected significantly by balloon dilatation. Stretched and torn fibers were observed frequently when 3 atm were applied. We concluded that the long-lasting effects of balloon dilatation may be caused by the disruption of connective tissues that proliferate in the vessel wall after a subarachnoid hemorrhage.

Accelerated non-muscle contraction after subarachnoid hemorrhage: cerebrospinal fluid testing in a culture model.

The cause of chronic cerebral vasospasm after subarachnoid hemorrhage has been studied intensively, but it is still controversial whether the observable luminal narrowing should be attributed to the contraction of vascular smooth muscle cells or whether it results from some organic change in the wall. A proliferation of myointimal cells, accompanied by increased deposition of collagen, as well as myonecrosis, have been frequently observed several days after aneurysm rupture. Studies from our laboratory showed that these myointimal cells had characteristics identical to myofibroblasts. In this study, we quantitatively and morphologically examined the effect of cerebrospinal fluid on the ability of myofibroblasts to alter collagen matrices using an in vitro model. Myofibroblasts contract the collagen matrix by rearranging or compacting the framework of collagen fibers. Cerebrospinal fluid obtained from patients with recently ruptured aneurysms significantly accelerated lattice contraction, especially when the patient developed symptomatic vasospasm. This study suggests that myofibroblasts in the spastic artery can produce a contractile force that contributes to chronic vasospasm, tightening the proliferated collagen. Some unknown agent present in bloody cerebrospinal fluid accelerates the rearrangement of the collagen lattice by myofibroblasts, both of which have, until now, been considered non-contractile components.

Cerebrospinal fluid factors following subarachnoid haemorrhage accelerate collagen lattice contraction by fibroblasts.

Proliferation in the intimal layer and medial necrosis are the most consistent findings in the cerebral artery following subarachnoid haemorrhage (SAH) in man. Recently, SEM studies from our laboratory have also shown marked endothelial injury as demonstrated by a profuse platelet carpet. Myofibroblasts proliferate in response to the platelet derived growth factor (PDGF), and abundant collagen is present in the vessel wall. We have employed experiments using fibroblast-populated collagen lattices to study cerebrospinal fluid (CSF) from patients with recent SAH. Isolated rat tail collagen and cultured human dermal fibroblasts are mixed together, placed in 35 mm Petri dishes, and allowed to gel. CSF samples are placed on the surface of the collagen lattice, using 0.2 ml saline for control. The collagen lattices are then incubated and daily measurements recorded. We found that CSF samples from patients with recently ruptured aneurysms significantly accelerate contraction of the collagen lattice. The factor in CSF is heat stable and has a molecular weight of less than 6000.

Physiological variables affecting collagen lattice contraction by human dermal fibroblasts.

Normal human dermal fibroblasts cultured in collagen lattices can compact that matrix by the process known as lattice contraction. That process is a model of the pathological one of scar contracture or wound contraction and is affected by several factors. Lattice contraction is promoted by the addition of adequate amounts of fetal bovine serum to the medium (maximum contraction with 10% serum). The process requires energy, of which glucose and pyruvate have been shown to be adequate sources. When glucose is used as the substrate, the major pathway of energy generation appears to be anaerobic metabolism. When pyruvate is the only substrate, aerobic metabolism may be crucial. The synthesis of DNA is not required for lattice contraction, while protein synthesis is, although the identities of the specific proteins are unknown. Impairment of calcium ion transport inhibits lattice contraction, and the specific inhibition of calmodulin-calcium interactions by W-7 blocks contraction. W-7 at a concentration of 6 x 10(-6) M blocks lattice contraction completely, while it has no effect at any lower concentration. Impairing dynamic microtubule activity impairs contraction. Disrupting microfilaments by cytochalasin B completely blocks lattice contraction. Microfilament function and calcium-calmodulin may be linked by a mechanism involving myosin-ATPase. The process of cell-mediated lattice contraction requires the production of energy, protein synthesis, and a functional cytoskeleton.

Morphologic examination of mesenchymal cells in healing wounds of normal and tight skin mice.

The healing process of an open wound as effected by wound contraction is complete by 3 weeks in the normal mouse. In contrast, its onset is delayed by 3 weeks and complete healing requires 6 weeks in the tight skin mouse (TSM), a mutant mouse strain with the autosomal dominant gene for tight skin. Possible mechanisms for this delay were evaluated. The frequency and distribution of myofibroblasts were studied during the 3-week delay in wound contraction by actin staining and electron microscopy. It was determined, by electron microscopy and phalloidin staining, that myofibroblasts were found in high density in noncontracting TSM wounds. Electron microscopy showed, however, that these myofibroblasts were surrounded by a pericellular matrix that separated their surface from adjacent collagen fibers. No pericellular matrix was found around cells in granulation tissue of normal mice. At 3 weeks, as TSM wounds began to contract, the number and intensity of cells stained by phalloidin in this tissue was less than that seen earlier. The pericellular matrix was fragmented at this time, and cell surface and collagen fiber associations were apparent. Finally, at 5 weeks, when wound contraction was well developed in the TSM, only a small area in the center of the healing wound beneath the epidermis contained phalloidin-positive myofibroblasts. Electron-microscopic examination of the residual granulation tissue at this time revealed the complete absence of the pericellular matrix. It is postulated that during the 3-week delay in wound closure, the presence of a localized pericellular matrix prevents the interaction between cells and collagen fibers necessary for the reorganization of collagen. It is also thought that the tightly adherent uninjured skin surrounding the healing wound may cause delayed wound closure. There was no evidence that the absence of myofibroblasts is responsible for delayed wound contraction.

Hyaluronate binding and degradation by cultured embryonic chick cardiac cushion and myocardial cells.

Cultured cells obtained from developing chick heart valvular and septal primordial tissues (cardiac cushions) and myocardium were tested for their capacity to bind, internalize, and degrade hyaluronate. A presumptive lysosomal hyaluronidase capable of hyaluronate degradation has been previously isolated and partially characterized from cultures enriched in either cushion tissue cells or myocardial cells (D. H. Bernanke and R. W. Orkin, 1984, Dev. Biol. 106, 351-359). In this study, both types of cultures were found to bind hyaluronate, but only the myocardial cultures could degrade the hyaluronate substrate. The lack of hyaluronate degradative capacity in the mesenchymal cushion tissue cells appears to result from their inability to internalize the macromolecule, thus failing to make it available to the lysosomal hyaluronidase. The data suggest that hyaluronate clearance from the extracellular matrix of the developing cushion is a complex process, involving more than simple extracellular degradation adjacent to the migrating mesenchymal cushion tissue cells. Instead, a sequence of events may be indicated which includes binding of hyaluronate to the cushion tissue cell surfaces and its transport by these cells across the cushion matrix toward the myocardium. The myocardium may be involved in the ultimate removal of hyaluronate from the cardiac jelly.

Hyaluronidase activity in embryonic chick heart muscle and cushion tissue and cells.

Hyaluronidase activity was compared in embryonic chick cardiac cushion and noncushion segments, as well as in cultures of mesenchyme derived from cardiac cushion endocardium (cushion tissue-enriched cultures) and in cultures of myocardial cells at stages critical to heart valve and septum development. Enzyme levels were higher in both heart tissue regions at periods of active cushion tissue mesenchyme migration than after migration ceases, and higher in the cushion region than in the noncushion region at both periods. Hyaluronidase was measured in cells and medium in both types of cultures, with five times greater activity found in the myocardial cultures. The cardiac hyaluronidase from cells and medium of both culture types had an estimated molecular weight of 41,000 to 44,000 and degraded hyaluronate and, to a lesser degree, chondroitin sulfate, at an acidic pH optimum. Ion-exchange chromatography demonstrated that in both culture types, a proportion of the secreted enzyme was more acidic than that found in the cell layer. These studies indicate the potential for hyaluronate degradation by the major cell types present in the developing heart at early stages and that the enzyme responsible is probably a lysosomal enzyme. Therefore, hyaluronate internalization is a likely requirement for degradation, and thus, the turnover of hyaluronate in developing heart valves is more complex than the extracellular degradative process suggested by histochemical data.

Effects of two glycosaminoglycans on seeding of cardiac cushion tissue cells into a collagen-lattice culture system.

A collagen-lattice culture model of developing heart valves was utilized to test two glycosaminoglycans, normally found in the cardiac jelly matrix of developing heart valve primordia, for their effects on the capability of mesenchymal derivatives of cardiac cushion endothelial cells to enter the substrate from the surface. Treatment with hyaluronate increased the rate of cell seeding to 2.04 times that of untreated control cultures and 1.82 times that of chondroitin sulfate-treated cultures. Scanning electron microscopic studies suggested that the increased rate was due to an enhanced disruption of intercellular junctions, influenced by hyaluronate, permitting disengagement of cells from the surface population and migration as mesenchymal cells into the collagen matrix. The results of this study correlate well with the presence of high hyaluronate concentrations in the cardiac jelly matrix beneath the cushion endothelium at periods of active seeding of cushion tissue cells.