David H. Bernanke

Development of the coronary blood supply: Changing concepts and current ideas

Earlier views of the development of the coronary vasculature included angiogenic budding and growth of arteries from the aortic sinuses and veins from the coronary sinus. The current concept begins with the establishment of the epicardium from the proepicardial organ, an outgrowth of the dorsal wall of the pericardial cavity. Capillaries form in a subepicardial mesenchymal population, extending as a plexus toward the truncus arteriosus and the atria. Multiple vessels grow from a peritruncal ring of capillaries, preferentially invading the newly formed aorta. In a process involving apoptotic changes of both the aortic wall and the invading capillaries, orifices open at the level of the aortic sinuses. Smooth muscle cells and pericytes, recruited from the surrounding mesenchyme, contribute to the vessel walls, and the definitive coronary artery pattern is established. Similar events are occurring on the venous side of the coronary circulation, following a slightly earlier time course. Multiple factors govern this process, including VEGF and FGF‐1 stimulating vasculogenesis and angiogenesis, and the angiopoietins and their tyrosine kinase receptors modulating interactions between endothelial cells and the mural components. As remodeling of the capillary plexus and the coronary orifices progresses, TGFβ released by apoptotic cells or from other sources likely modulates VEGF and FGF‐1, and also contributes to further apoptotic changes. A better appreciation of the controls of the mechanisms of coronary vessel development may direct further research in the prevention of arteriosclerosis and ischemic tissue injuries. Anat Rec (New Anat) 269:198–208, 2002.

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.

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.

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. Anat Rec 262:310–317, 2001.

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 Gomoris 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.

Stroke: anatomy of a catastrophic event.

Subarachnoid hemorrhage (SAH) resulting from the rupture of a cerebral aneurysm represents one major cause of stroke. SAH may be followed by a spontaneous severe contraction of major cerebral arteries, a condition referred to as cerebral vasospasm. Vasospasm may result in brain ischemia or actual tissue death. This constrictive vascular state is devastating, remains largely untreatable, and is a major cause of morbidity and mortality in SAH patients. Approximately 30,000 Americans are affected by this condition each year. The overall death rates are 25%, and significant neurological complications occur in 50% of individuals who survive the initial bleed. This report highlights some of the important aspects of this vascular disease. Anat. Rec. (New Anat.) 253:58‐63, 1998.

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.

Angioplasty of vasospasm: Is it reasonable?

Clinical and radiographic examinations indicate preliminarily indications that transluminal angioplasty may be effective in overall management of the patient with vasospasm. Many questions remain, including: How does it work?; Are the effects persistent?; Is the arterial wall injured by the process? Recent studies in several patients who died after angioplasty allow us to provide some answers. Undilated spastic arteries show proliferation of both cellular and connective tissue elements. There is good evidence that myofibroblasts have reorganized the collagen framework, increasing fibril density and thus thickness. Dilated vessels show thinning of the arterial wall without disruption but with compaction of the new collagen fibrils. Cellular nests are also compressed and stretched. The endothelial layers are undisturbed. The success of dilatation depends on the amount and location of proliferation present. The effect is usually permanent. Because our protocols call for dilatation no greater than 10% above normal diameter, the muscle layers have not been torn or stretched although focal areas of necrosis are sometimes seen. Understanding the constrictive process and its relief through dilatation, allows us to formulate a therapeutic plan. Our experience in treating 89 patients with vasospasm after SAH suggests that, for best results, angioplasty should be performed before the angiopathic features become florid. This helps to preserve flow through the short arteries to the brain stem and deep brain nuclei, which may be involved indirectly in the vasospastic process.

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.