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- MATERIALS AND METHODS
- LITERATURE CITED
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. © 2001 Wiley-Liss, Inc.
Detailed time-course studies of coronary artery development in the quail (Bogers et al., 1988, 1989) and chicken (Waldo et al., 1990) have established that the proximal segments of coronary arteries develop via the growth of vessels from the peritruncal capillary plexus into the truncus arteriosus. As the truncus arteriosus undergoes septation to form the aortic and pulmonary outflow tracts, vascular channels arising from the peritruncal capillary plexus begin to extend into the aortic mesenchyme. Vessels from the peritruncal capillary plexus contact the aortic endothelium and form presumptive coronary orifices in the aortic sinuses. Waldo et al. (1990) noted that vessels from the peritruncal capillary plexus did not invade the pulmonary artery, and they observed multiple connections to the left, right, and, rarely, posterior aortic sinuses in the early development of the proximal coronary artery segments.
Waldo et al. (1990) also found that the multiple connections persisted in the left and sometimes right aortic sinuses, but not in other aortic sites. Studies in the adult human heart (Vlodaver et al., 1975; Angelini, 1989; Turner and Navaratnam, 1996) similarly described the rather frequent occurrence (≈35%) of small accessory coronary orifices in the left and right aortic sinuses and the relative rarity (0.5%) of coronary orifices in the pulmonary artery or other aortic sites. Bogers et al. (1989) did not observe multiple channels, but they too observed that vessels from the peritruncal capillary plexus penetrated the left and right aortic sinuses in preference to other aortic sites and the pulmonary artery.
After the capillaries from the peritruncal plexus connect to the aorta, rapid remodeling of the vessels and their orifices follows. By an as yet unknown mechanism, some of the penetrating vessels acquire a tunica media (vascular smooth muscle layer) and persist to become the definitive coronary arteries (Hood and Rosenquist, 1992; Poelmann et al., 1993; Waldo et al., 1994; Vranken-Peeters et al., 1997). Poelmann et al. (1993) observed that other penetrating vessels fail to form a tunica media and subsequently regress, and they postulated that the acquisition of a tunica media plays a stabilizing role in coronary artery development. Other investigators have suggested that parasympathetic ganglia derived from cardiac neural crest cells, although not directly contributing to coronary vessel elements, significantly affect the organization and development of coronary arteries (Hood and Rosenquist, 1992; Waldo et al., 1994). Beyond these findings, however, the exact mechanism directing the pattern development of the proximal coronary arteries is unknown.
Similarly, the factors that mediate the initial ingrowth of vessels from the peritruncal capillary plexus into the aorta remain a mystery. It is also unclear why vessels from the peritruncal capillary plexus bypass the pulmonary artery and selectively penetrate the aorta. The specificity for coronary orifice formation within the left and right aortic sinuses, however, indicates the presence of some regulatory mechanisms during their development. In fact, Waldo et al. (1990) drew the specific conclusion that the process by which vessels from the peritruncal capillary plexus penetrate the aortic sinuses is an angiogenic event that “represents a controlled invasion of the aorta.”
This conclusion by Waldo et al. (1990) is not without significance, because the process of angiogenesis in general has been described as the regulated invasion of one tissue by cells from another tissue (Moscatelli and Rifkin, 1988) requiring rather complex cell to cell signaling (Joseph-Silverstein and Rifkin, 1987). In the case of coronary artery development, additional complexity exists beyond merely penetrating the aorta. Upon contacting the aortic endothelium, some process must ensue by which a patent connection between the penetrating vessels and the aorta can be made. With further development of the coronary arteries, there is also the need for the aorta to somehow accommodate the expanding coronary orifice. Finally, as the definitive coronary arteries are elaborated, extensive remodeling of the coronary vascular plexus must follow as some channels are incorporated into the arterial system although others regress (Vranken-Peeters et al., 1997). Thus, development of the proximal coronary arteries and their orifices requires a drastic and rapid reorganization of many tissues, and the purpose of this study was to elucidate the mechanical events involved during this process.
It stands to reason that the development of the proximal coronary arteries and their orifices requires the displacement or removal of cells. The present study explores the hypothesis that apoptosis is involved in this process. Apoptosis or programmed cell death is a highly regulated event that can be induced or inhibited by a variety of extracellular and intracellular signals, that have been well characterized (Huppertz et al., 1999). Previous investigators have noted the presence of apoptotic cells during embryonic development (Jacobson et al., 1997) and in embryonic tissues undergoing remodeling such as the nervous system (Raff et al., 1993), distal appendages (Garcia-Martinez et al., 1993), and the heart (Pexieder, 1975; Icardo, 1990). While examining presumed coronary artery sprouting, Aikawa and Kawano (1982) noted the presence of irregular and apparently degenerating endothelial cells in the aortic wall at or near the aortic sinuses. Recent studies specifically investigating outflow tract development have demonstrated that apoptosis is associated with outflow tract shortening (Watanabe et al., 1998) and septation (Poelmann et al., 1998). The present study uses established histochemical and ultrastructural techniques to determine if apoptotic cells are present in sites of the aorta associated with coronary artery orifice formation.
- Top of page
- MATERIALS AND METHODS
- LITERATURE CITED
The concept of coronary ingrowth has been well established by numerous studies (Bogers et al., 1989; Waldo et al., 1990; Hood and Rosenquist, 1992; Poelmann et al., 1993; Waldo et al., 1994), but an understanding of the mechanisms regulating this process has remained elusive. Previous studies have suggested the importance of cardiac neural crest derived cells in the aorticopulmonary septum (Hood and Rosenquist, 1992) and parasympathetic ganglia (Waldo et al., 1994) for the development of coronary arteries. Yet, in a recent review of the subject, Tomanek (1996) emphasized that “Our knowledge of the regulation of coronary vascularization is very limited. Accordingly, future studies need to focus on the role of growth factors, chemotactic factors, extracellular matrix molecules, and mechanical events.” The present study has focused upon the “mechanical events” involved in coronary artery orifice formation. We believe our findings, as summarized in Figure 4, refine the model of coronary ingrowth proposed by Bogers et al. (1989) and Waldo et al. (1990). Apoptotic cells were observed in close spatial and temporal association with the developing coronary arteries and their orifices. During the invasion of vessels from the peritruncal capillary plexus into the aorta, apoptotic cells were found in association with the peritruncal capillary plexus but not within the endothelium of the aortic sinuses. At the time of coronary vessel connection to the aorta (HH 32–33), however, numerous apoptotic cells were seen within the lumens of the coronary orifices and along the margins of the vessels. Even after the coronary arteries had well-established connections to the aorta (HH 34–35), apoptotic cells were still found at the sites of the expanding coronary orifices and along the margins of the developing coronary arteries.
Figure 4. A model of coronary artery orifice formation. A: Invasion. During the process of vascular invasion of the aorta, blind-ended vessels (PTV) from the peritruncal capillary plexus invade the aortic tissue (TM). Apoptotic cells (Apo) are found in association with the proliferating vessels of the peritruncal capillary plexus, but not within the aortic endothelium (En). B: Connection. When the invading vessel contacts the aortic endothelium, the interface between the aortic endothelium and the blind end of the vessel is transformed into a patent orifice via apoptosis. C: Remodeling. After connecting to the aorta, some of the penetrating vessels are selected by an as yet unknown mechanism to develop into the definitive proximal coronary arteries. These vessels begin to acquire a vascular smooth muscle coat (VSMC) and increase in diameter. The coronary orifice and the surrounding aortic tissue must accommodate the expansion of the coronary arteries, and it is presumed that apoptosis plays a vital role in this remodeling process.
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These observations suggest that: 1) the process by which the nascent coronary arteries invade and form patent connections to the aorta involves apoptosis; and 2) subsequent remodeling of the coronary arteries and their orifices involves apoptosis. These findings are encouraging because the apoptosis cascade, as recently reviewed by Huppertz et al. (1999), is itself a process tightly regulated by extracellular and intracellular signals about which there is a vast, and still growing, body of knowledge. As such, the correlation of apoptosis with coronary artery orifice formation sheds new light on the possible regulatory mechanisms involved.
In the context of frequent observations of accessory coronary orifices in adults (Vlodaver et al., 1975, Angelini, 1989, Turner and Navaratnam, 1996) and the direct observation of multiple connections of coronary vessels to the aorta in embryos (Waldo et al., 1990; Hood and Rosenquist, 1992; Poelmann et al., 1993; Waldo et al., 1994), it seems reasonable to presume that the invading vessels from the peritruncal capillary plexus might have a locally inductive effect on aortic tissue. That is, as vessels from the peritruncal capillary plexus invade the aorta and contact the aortic endothelium, the proliferating vessels might, in some way, induce apoptosis within the aortic mesenchyme and endothelium. The frequency with which we observed mitotic cells in the perivascular regions where we also detected apoptotic cells suggests an interaction between vascular proliferation and apoptosis that certainly deserves further attention.
The fact, however, that vessels from the peritruncal capillary plexus surround both the aorta and the pulmonary artery, yet generally only invade the aorta suggests a chemotactic mechanism that is as yet unknown. The observation of transient connections to the posterior (non-coronary) aortic sinus in embryos (Waldo et al., 1990; Hood and Rosenquist, 1992; Poelmann et al., 1993; Waldo et al., 1994) that do not survive in later stages suggests that any local inductive effect of invading vessels from the peritruncal capillary plexus does not necessarily determine the ultimate organization of the mature coronary arteries. Furthermore, the possibility that the apoptosis observed in this study precedes and quite possibly induces vascular invasion of the aorta should be considered. Thus, it is not clear if the apoptosis we observed is a cause or an effect of the ingrowth, connection, and remodeling of nascent coronary arteries to the aorta. Clearly, more research is required regarding the exact timing and regulation of the apoptosis to determine whether the apoptotic cells are “inductors” or “inductees” in the process of coronary artery development.
It must also be emphasized that although apoptotic cells were detected in the area of the developing coronary arteries and their orifices, these cells have not been characterized. It is not known if the apoptotic cells observed are derived from local (aortic) mesoderm, cardiac neural crest, nascent coronary vessel elements, or a combination thereof. Similarly, it remains to be determined whether specific signaling molecules already known to be involved in angiogenesis, apoptosis, and differentiation are present (or absent) during the process of proximal coronary artery development and orifice formation.
The results of this study do not clearly indicate any one specific regulatory mechanism at work during coronary artery development. In the absence of any characterization of the apoptotic cells or any experimental manipulation of aortic and coronary vascular tissue, it is difficult to offer a molecular explanation for the complex events involved in coronary artery development. The results of this study only confirm that apoptosis is involved in the formation of the proximal coronary arteries and their orifices. These findings generate avenues for future research and suggest that factors involved in regulating apoptosis should be included in future models of coronary artery development.