Robert J. Tomanek

Angiogenesis: New insights and therapeutic potential

Angiogenesis, the formation of vessels from pre‐existing vessels, is of critical importance not only during normal growth, but also in pathological situations. In the latter, some diseases are enhanced by excessive vascular growth (e.g., tumors), whereas in others inadequate vascular growth contributes to morbidity and mortality (e.g., ischemic heart disease). Our current state of knowledge makes it clear that the cascade of angiogenic events depends on complex processes that include cell‐cell interactions, various intracellular signaling pathways, and the appropriate extracellular microenvironment. The literature regarding angiogenesis has increased exponentially during the last decade. Progress in this area is largely a consequence of advances in our understanding of angiogenic growth factor and cytokine function, in part due to the determination of their complete amino acid sequences and cloning of their genes. Other factors also play key roles in angiogenesis, including the extracellular matrix, adhesion molecules and their inhibitors, and metabolic and mechanical factors. The potential for developing therapeutic protocols has been enhanced by data from both in vitro and in vivo studies and has provided the rationale for clinic trials. Angiogenic therapy strategies include inhibition of aberrant angiogenesis, as seen in tumors or diabetes, as well as stimulation of angiogenesis in conditions of ischemia, such as ischemic heart or peripheral vascular disease. Anat Rec (New Anat) 261:126–135, 2000.

Effects of duration and severity of arterial hypertension and cardiac hypertrophy on coronary vasodilator reserve.

Cardiac hypertrophy is associated with a decrease in coronary reserve. However, factors which may modulate the interaction between myocardial growth and vascular proliferation, such as duration and severity of hypertrophy, have not been evaluated. We measured myocardial perfusion with microspheres in conscious, chronically instrumented Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats at 3, 7, and 15 months of age; and in SHR stroke-prone (SHR-SP) rats at 13–14 months of age. Myocardial perfusion was measured with microspheres in awake rats at rest and during maximal coronary dilation produced by dipyridamole infusion (2.0 mg/kg per min, iv). Arterial pressure was significantly elevated (P < 0.05) in all hypertensive groups (vs. age-matched WKY), both at rest and during dipyridamole infusion. Left ventricular mass in the SHR rats was increased significantly (P < 0.05) by 14%, 28%, and 29% at 3, 7, and 15 months, respectively. Left ventricular mass in the SHR-SP group was increased by 50% (P S 0.05) compared to the 15-month-old WKY. Left ventricular minimal coronary vascular resistance (per gram) was significantly greater (P < 0.05) in SHR at 7 months, and in the SHR-SP group (66% and 60%, respectively). Right ventricular minimal coronary vascular resistance was significantly greater (P < 0.05) in SHR at 7 and 15 months (50%), and in the SHR-SP group (122%), compared to 15-monthold WKY. The results indicate the following: (1) the increase in minimal coronary vascular resistance between SHR and WKY rats was greatest when left ventricular hypertrophy peaked (7 months) and was no longer present after left ventricular hypertrophy had stablized. (2) In 14-month-old SHR-SP rats, with more severe left ventricular hypertrophy and hypertension, minimal coronary vascular resistance was considerably higher than in SHR of approximately the same age. (3) Long-term arterial hypertension was associated with a higher right ventricular minimal coronary vascular resistance. Resistance appeared to change in proportion to the severity of hypertension, and the changes were independent of the presence of right ventricular hypertrophy.

Response of the coronary vasculature to myocardial hypertrophy

Cardiac hypertrophy is often characterized by abnormalities in myocardial perfusion, including decreased coronary reserve, increased minimal coronary vascular resistance, underperfusion of the subendomyocardium during conditions of high oxygen demand and increased risk of infarction in the presence of coronary occlusion. Two major anatomic variables may cause these perfusion deficits. First, the coronary resistance vessels may not grow in proportion to the magnitude of the cardiac enlargement. Second, the luminal diameter of resistance vessels may become reduced as a consequence of medial hypertrophy, hyperplasia or fibrosis. A luminal narrowing coupled with a lack or inadequate numeric proliferation of resistance vessels can markedly limit maximal myocardial perfusion. However, not all models of cardiac hypertrophy are characterized by perfusion abnormalities. A substantial growth of arterioles and capillaries has been documented in exercise- and thyroxine-induced left ventricular hypertrophy. Moreover, at least in some models, angiogenesis occurs when the duration of the ventricular hypertrophy is sufficient. The hypothesis that coronary angiogenesis is stimulated by increased blood flow or prolongation of diastole appears to have support from a number of experimental studies. However, the cascade of events underlying angiogenesis and the numerous variables that characterize the various models of hypertrophy are complex and require elucidation.

Quantitative changes in the capillary bed during developing, peak, and stabilized cardiac hypertrophy in the spontaneously hypertensive rat.

We studied the effects of developing, peak, and stabilized cardiac hypertrophy on the capillary bed of spontaneously hypertensive rats; Wistar-Kyoto rats served as controls. Histological measurements were based on 1.5-/xm cross-sections of left ventricular specimens from perfuse-fixed hearts arrested in diastole. The decline in capillary density, in general, paralleled the decrease in capillary surface area which was lowest at peak hypertrophy (7 months) in spontaneously hypertensive rats. Between 7 and 15 months cardiocyte diameter remained constant and capillary density increased to Wistar-Kyoto rat values. Radioautographic data showed that in 12-month-old spontaneously hypertensive rats, 3H-thymidine labeling of capillary nuclei was more than 2-fold higher than in Wistar-Kyoto rats. Mean capillary diameter increased in both spontaneously hypertensive and Wistar-Kyoto rats between 1 and 2.5 months, but remained constant thereafter. Whereas multiple regression analysis showed that capillary density is the major determinant of capillary surface area, significant changes were detected earlier in capillary surface area than in capillary density. Although peak cardiac hypertrophy affected a 12.5% capillary density decrement in spontaneously hypertensive rats, capillary surface area was 24% lower than in Wistar-Kyoto rats. This substantial decrease in capillary surface area was associated with only a 1.1-jU.m decrease in minimal intercapillary distance. Since mean capillary diameter was similar in the two strains, it is suggested that the drop in capillary surface area during peak hypertrophy was due to a relative decrease in anastomotic and branching capillaries, as well as the decline in capillary density. These data provide morphological evidence that: (1) whereas capillary density is, in general, a good predictor of capillary surface area, the latter is a more sensitive measure of capillarity than the former; (2) the decrement in capillary supply to a cardiocyte during peak, but moderate, hypertrophy is probably substantially greater than that estimated by capillary density; and (3) capillary growth during stabilized hypertrophy is sufficient to reverse the decrements in capillary surface area, capillary density and the increase in minimal intercapillary distance.

Vascular endothelial growth factor expression coincides with coronary vasculogenesis and angiogenesis.

Vascular endothelial growth factor (VEGF) plays an important role in early embryonic vasculogenesis. To establish its temporal expression and localization in the heart during development, we studied rat hearts from the first embryonic day (E) of myocardial vascular tube formation through the early postnatal period. Ventricular VEGF immunoreactivity was noted in the epicardium and the thin underlying myocardium in E10 ventricles. During the earliest stages of vascularization (E13–E16) immunoreactivity was highest in the compact myocardium nearest the epicardium, and subsequently (E18 and thereafter) became more evenly distributed transmurally. By birth (E22) immunoreactivity was most intense around microvessels. Similarly, VEGF mRNA localization, demonstrated by in situ hybridization, was initially highest near the epicardium and then became more evenly distributed transmurally by late gestation. Within the interventricular septum, the highest expression occurred in the middle of the wall where it correlated with the greatest vascularization. Northern blot analysis showed that from E12 through the first 10 days of postnatal life, VEGF was two to three times higher than in the adult. Western blot analysis showed that VEGF tended to be higher in the atria than the ventricles, and negligible in the outflow tract. Our data indicate that VEGF localization and expression 1) correspond to the pattern of vascularization in the embryonic/fetal heart, and 2) remain high during the early postnatal period when capillary proliferation is high. Because VEGF is stimulated by hypoxia, its preferential mRNA expression near the epicardium, that is, farthest from the ventricular lumen and the O2 source, fits with the hypothesis that a hypoxic gradient is a driving force in the transmural vascularization process. Dev Dyn 1999;215:54–61.

Formation of the coronary vasculature during development.

The formation of the coronary vasculature involves a series of carefully regulated temporal events that include vasculogenesis, angiogenesis, arteriogenesis and remodeling. This review explores these events, which begin with the migration of proepicardial cells to form the epicardium and end with postnatal growth and remodeling. Coronary endothelial, smooth muscle and fibroblast cells differentiate via epithelial–mesenchymal transformation; these cells delaminate from the epicardium. Following the formation of a tubular network by endothelial cells, an aortic ring of endothelial cells penetrates the aorta at the left and right aortic cusps to form the two ostia. Smooth muscle cell recruitment occurs rapidly and the coronary artery network begins forming as blood flow is established. Recent studies have identified a number of regulatory molecules that play key roles in epicardial formation and the transformation of its component cells into mesenchyme. Moreover, we are finally gaining some understanding regarding the interplay of angiogenic growth factors in the complex process of establishing the coronary vascular tree. Understanding coronary embryogenesis is important for interventions regarding adult cardiovascular diseases as well as those necessary to correct congenital defects.

Myocardial ultrastructure of young and senescent rats

Myocardial ultrastructure was compared in specimens from rats aged 3–6 months (young) and 27–28 months (senescent). Retrograde aortic aldehyde perfusion produced uniform preservation. Characteristic effects were observed with variations in buffer systems, fixative concentration, and osmolality. Myocardial cells of senescent rats are characterized by increased lipofuscin pigment containing material of varying density; the granules are often associated with smaller lysosomelike structures. Golgi structures are numerous. Foci of small densely accumulated mitochondria, and dilated vesicles in the intercalated disc region are frequent. Irregular myofibers, numerous surface vesicles, and increased numbers of connective tissue cells are common. These findings suggest that aging of the myocardium includes structural alterations which are not altogether consistent with various pathological conditions.

Differential Healing Activities of CD34+ and CD14+ Endothelial Cell Progenitors

Objective—Peripheral blood contains primitive (stem cell-like) and monocytic-like endothelial cell progenitors. Diabetes apparently converts these primitive progenitors, from a pro-angiogenic to anti-angiogenic phenotype. Monocytic progenitors seem to be less affected by diabetes, but potential pro-angiogenic activities of freshly isolated monocytic progenitors remain unexplored. We compared the ability of primitive and monocytic endothelial cell progenitors to stimulate vascular growth and healing in diabetes and investigated potential molecular mechanisms through which the cells mediate their in vivo effects. Methods and Results—Human CD34+ primitive progenitors and CD14+ monocytic progenitors were injected locally into the ischemic limbs of diabetic mice. CD14+ cell therapy improved healing and vessel growth, although not as rapidly or effectively as CD34+ cell treatment. Western blot analysis revealed that cell therapy modulated expression of molecules in the VEGF, MCP-1, and angiopoietin pathways. Conclusions—Injection of freshly isolated circulating CD14+ cells improves healing and vascular growth indicating their potential for use in acute clinical settings. Importantly, CD14+ cells could provide a therapeutic option for people with diabetes, the function of whose CD34+ cells may be compromised. At least some progenitor-induced healing probably is mediated through increased sensitivity to VEGF and increases in MCP-1, and possibly modulation of angiopoietins.

Chronic progressive pressure overload of the cat right ventricle.

When an abrupt, fixed increase in afterload induces hypertrophy, the myocardium exhibits normal pump function in vivo, depressed muscle function in vitro, and paradoxically increased oxygen consumption. Such abnormalities may be either a transient response to a reversible myocardial injury or instead may be a persistent characteristic of hypertrophy contributing to eventual heart failure. To distinguish between these alternatives, we developed a model of chronic progressive pressure overload. Kittens had either a sham operation or pulmonary banding for 25 (group I) or 60 (group II) weeks. Banding produced a gradually increasing pressure overload with growth, maximum at 16 weeks after operation. The ratio of right ventricular to body weight increased from 0.54 ± 0.03 to 0.82 ± 0.04 g/kg (P < 0.01) in group I and from 0.50 ± 0.03 to 0.72 ± 0.03 (P < 0.01) in group II. In vivo right ventricular pump function (cardiac output and ejection fraction) was normal in both groups. In vitro contractile function and metabolism were measured in papillary muscles from the same right ventricles. Both experimental groups showed marked contractile abnormalities: preloaded shortening velocity was reduced from 0.80 ± 0.05 to 0.53 ± 0.05 muscle length/sec (P < 0.01) in group I and from 0.84 ± 0.04 to 0.60 ± 0.06 (P < 0.01) in group II. Maximum isometric active tension was reduced from 60 ± 7 to 35 ± 5 m N / W (P< 0.01) in group Iand from 56 ± 5 to 27 ± 4 (P< 0.01) in group II. Metabolism was normal in both experimental groups. Thus, under conditions relevant to the study of clinical cardiac hypertrophy that are unlikely to cause acute injury, hypertrophy produces persistently abnormal intrinsic contractile function. Circ Res 48: 488-497, 1981

Bradycardia-Induced Coronary Angiogenesis Is Dependent on Vascular Endothelial Growth Factor

A marked coronary angiogenesis is known to occur with chronic bradycardia. We tested the hypothesis that vascular endothelial growth factor (VEGF), an endothelial cell mitogen and a major regulator of angiogenesis, is upregulated in response to low heart rate and consequential increased stroke volume. Bradycardia was induced in rats by administering the bradycardic drug alinidine (3 mg/kg body weight) twice daily. Heart rate decreased by 32% for 20 to 40 minutes after injection and was chronically reduced by 10%, 14%, and 18.5% after 1, 2, and 3 weeks of treatment, respectively. Arterial pressure and cardiac output were unchanged. Left ventricular capillary length density (mm/mm(3)) increased gradually with alinidine administration; a 15% increase after 2 weeks and a 40% increase after 3 weeks of alinidine treatment were documented. Left ventricular weight, body weight, and their ratio were not significantly altered by alinidine treatment. After 1 week of treatment, before an increase in capillary length density, VEGF mRNA increased >2-fold and then declined to control levels after 3 weeks of treatment. VEGF protein was higher in alinidine-treated rats than in controls after 2 weeks and increased further after 3 weeks of treatment. Injection of VEGF-neutralizing antibodies over a 2-week period completely blocked alinidine-stimulated angiogenesis. In contrast, bFGF mRNA was not altered by alinidine treatment. These data suggest that VEGF plays a key role in the angiogenic response that occurs with chronic bradycardia. The mechanism underlying this VEGF-associated angiogenesis may be an increase in stretch due to enhanced diastolic filling.