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Keywords:

  • coronary artery development;
  • coronary orifice development;
  • heart development;
  • chick embryo;
  • apoptosis

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. 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.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Light Microscopy

White Leghorn chicken eggs (Tyson Foods, Magee, MS) were incubated at 37°C and at 70% humidity for 6–9 days. After careful staging according to criteria established by Hamburger and Hamilton (1951), the embryos were decapitated and divided at the level of the diaphragm. Thoracic segments were collected and fixed in 0.1 M phosphate buffered 4% paraformaldehyde at 4°C for 2 hr. After fixation, specimens were rinsed in 0.1 M phosphate buffer, dehydrated in ethanol, embedded in paraffin, and sectioned in the coronal plane at 10 μm. Serial sections were mounted on Superfrost plus slides (Fisher Scientific, Pittsburgh, PA) and routinely stained with hematoxylin and eosin. The specimens were examined and photographed using an Olympus IMT-2B inverted microscope. Digital images of specimens were captured using a Leica Leitz DMRX microscope equipped with a 1.2 megapixel SPOT CCD camera (Diagnostic Instruments, Sterling Heights, MI).

TUNEL Staining for Apoptosis

A commercially available terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) kit (Roche Molecular Biochemicals, Indianapolis, IN) was used to detect apoptotic cells. HH 29–35 embryos were obtained as described above and fixed in 0.1 M phosphate buffered 4% paraformaldehyde at 4°C for 2 hr, followed by dehydration, embedding, and serial sectioning. The sections were deparaffinized, rehydrated, and evaluated with an Olympus IMT-2B inverted microscope equipped with Hoffman modulation contrast optics. Sections demonstrating the presence of left and right aortic sinuses were selected for TUNEL staining and ringed with a PAP pen (Research Products International, Mount Prospect, IL). Selected sections were rinsed in 0.1 M. pH 7.6 Tris HCL-buffered saline (TBS), treated for 15 min at 37°C in a humidity chamber with proteinase K (20 μg/ml) in 10 mM Tris HCL, pH 7.6, and rinsed twice with TBS. The sections were treated with TUNEL reactants for 1 hr at 37°C in a humidity chamber followed by two rinses with TBS. After the TUNEL reaction, sections were evaluated again with Hoffman modulation contrast optics to ensure that tissue processing did not alter the morphological features of the specimens. After examination with Hoffman optics, the specimens were evaluated and photographed using an epifluorescence microscope (Olympus IMT-2B) equipped with a fluorescein filter. Appropriate positive and negative controls were included in the TUNEL assay. As an internal control, the presence of apoptotic cells was verified in the outflow tract as previously described by Watanabe et al. (1998) and Poelmann et al. (1998).

Transmission Electron Microscopy

HH 29–35 embryos were collected as described above and the hearts dissected free. The hearts were immersed in modified Karnovsky's fixative (4% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M, pH 7.4 phosphate buffer) at 4°C for 2 hr, rinsed with 0.1 M phosphate buffer, and then post-fixed with 1% osmium tetraoxide in 0.1 M pH 7.4 phosphate buffer. The hearts were stained en bloc with aqueous uranyl acetate (2%) and then embedded in Epon resin. Survey sections in the coronal plane were cut at 1 μm, mounted on glass sides, stained with 1% toluidine blue in 1% sodium borate, and examined with a light microscope. Survey sections were cut until the left and right aortic sinuses could be observed. The specimen blocks were trimmed around the area of the aortic sinuses, and 60 nm sections were cut using a Reichert-Jung ultramicrotome. Serial sections were collected on 100 mesh hexagon grids or formvar-coated slotted grids and stained with aqueous lead citrate (2%) and aqueous uranyl acetate (2%). Sections were examined and photographed using a LEO 906 transmission electron microscope.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

Capillary Invasion Into the Aortic Media

Light microscopy.

Consistent with the previous findings of Bogers et al. (1989) and Waldo et al. (1990), no coronary artery orifices were observed in any specimens before stage HH 32 (Fig. 1a). By HH 31, an elongated cuff of myocardial tissue was apparent that extended to the level of the developing aortic semilunar valve, as was a somewhat disorganized aortic media, and numerous vascular channels, that were assumed to be part of the peritruncal capillary plexus surrounding the pulmonary artery and aorta. Although some of the channels very closely approximated the aortic lumen, serial sections revealed a continuous aortic endothelium without any aortic evagination.

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Figure 1. Vascular invasion of the aorta. A: Routine H and E stained section of an HH 29 heart showing the aortic lumen (AO), leaflets (VL) of the developing aortic semilunar valve, and the outflow portion of the left ventricle (VTR). Several cross-section profiles of vessels within the peritruncal capillary plexus (PTCP) can be seen surrounding the aorta. There is no obvious outgrowth of the aorta toward the PTCP (×150). B: TUNEL stained section of an HH 29 heart showing similar morphological features as in Figure 1A. TUNEL positive cells (white arrow) can be seen within the myocardial cuff (MC). TUNEL positive cells are also found in areas around the PTCP, but not within the aortic endothelium (×150). C: TEM micrograph showing the invasion of aortic tunica media (TM) by a vessel (PTV) from the peritruncal capillary plexus. The micrograph also shows an apparent deflection (DEF) of the aortic endothelium toward the invading vessel. The endothelial cells within the deflection (black arrowhead) are atypically stellate rather than squamous. An area of disrupted aortic tissue (DIS) immediately surrounding the invading vessel displays higher cell density and different cellular morphology compared to other regions of the tunica media. A mitotic figure (*) is seen within the disrupted tissue (×1,100).

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TUNEL staining.

TUNEL staining in the aortas of HH 29–31 embryos (Fig. 1b) revealed specific sites of TUNEL positive cells. As previously described, numerous TUNEL positive cells were detected within the myocardial cuff extending to the level of the aortic valve leaflets (Watanabe et al., 1998) and within the aorticopulmonary septum (Poelmann et al., 1998). TUNEL positive cells were also found within the developing valve leaflets. Most notably, TUNEL positive cells were consistently observed in the areas of the aorta that most closely approximated the peritruncal capillary plexus. The aortic endothelium and the innermost layers of the aortic media, however, had very few or no TUNEL positive cells at these stages.

Transmission electron microscopy.

As with the LM survey of aortas in HH 29–31 embryos, TEM examination of HH 29–31 hearts did not reveal any pronounced outgrowth of the aorta toward penetrating vessels from the peritruncal capillary plexus (Fig. 1c). These penetrating vessels were blind ended, endothelial-lined channels that contained erythrocytes, and, thus, could be readily identified within the aortic media. In instances where a penetrating vessel from the peritruncal capillary plexus very closely approximated the aortic endothelium, a slight deflection (≈30 μm) of the aortic endothelium toward the penetrating vessel was commonly observed. The cells of the deflected aortic endothelium had an atypically ovoid appearance. The normally lamellar organization of the aortic media appeared disrupted in the areas surrounding penetrating vessels from the peritruncal capillary plexus. The cells of these areas were irregularly shaped and rather densely packed as compared to other regions of the aorta.

Connection to the Aorta

Light microscopy.

In HH 32 and HH 33 embryos (Fig. 2a), a shortened myocardial cuff was found along with well-formed aortic valve leaflets, and dilated aortic sinuses. The media of the aorta seemed better organized, and numerous examples of coronary artery orifices were observed in the left and right aortic sinuses that were continuous with a penetrating vessel from the peritruncal capillary plexus. The penetrating vessels were about 15–20 μm in diameter and seemed to consist of little more than an endothelium. A left coronary artery orifice was usually seen before the appearance of a right orifice as previously described by Bogers et al. (1989). Serial sections also revealed multiple vascular channels connecting to the left and right aortic sinuses in agreement with the findings of Waldo et al. (1990). No coronary artery orifices were found in either the pulmonary artery or the posterior aortic sinus.

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Figure 2. Coronary artery orifice formation. A: Routine H and E stained section of an HH 33 heart showing the aortic lumen (AO), leaflets (VL) of the aortic semilunar valve, and the aortic sinuses (AS). A coronary orifice (white arrow) is observed which, when viewed in serial sections (not shown), is continuous with the developing coronary artery (black arrow) (×150). B: TUNEL stained section of an HH 33 heart showing aorta (AO), valve leaflets (VL), and the left aortic sinus (AS). A coronary orifice (black arrow) is observed that is continuous with the developing coronary artery (CA). TUNEL positive cells (white arrows) are detected within the myocardial cuff (MC) and, most notably, along the margins of the developing coronary artery (×150). C: TEM micrograph of an HH 33 heart showing the lumen of a coronary artery orifice (CO) and a coronary artery (CA). Numerous apoptotic cells (Apo) may be seen within the lumen. An aggregation of endothelial cells (En) is observed protruding into the lumen. A mitotic figure (*) is observed immediately underneath the aggregation of cells (×1,400). D: A higher magnification TEM micrograph of a cell observed within the lumen of another coronary orifice shows the ultrastructural characteristics of apoptosis such as cytoplasmic blebbing (B) and annular chromatin condensation (CC) within a still intact nucleus (Nuc) (×13,200).

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TUNEL staining.

TUNEL staining in the aortas of HH 32–33 embryos (Fig. 2b) demonstrated positive cells in the myocardial cuff, although the overall number of positive cells within the cuff had decreased dramatically compared to prior stages. TUNEL positive cells could also be found within the valve leaflets. Labeled cells were still observed in association with the peritruncal capillary plexus. Most notably, TUNEL positive cells were found along the aspects of penetrating coronary vessels between the peritruncal capillary plexus and the sites of their attachment to the aorta. There were frequent instances of positive cells within the coronary artery orifice itself.

Transmission electron microscopy.

At the coronary artery orifices in HH 32–33 embryos, numerous cells exhibiting morphological characteristics of apoptosis were found within the lumen of the orifice (Fig. 2c,d). Cytoplasmic fragments suggestive of cell blebbing and cells exhibiting extensive chromatin condensation were observed within the lumens of several coronary artery orifices. As with the HH 29–31 embryos, the lamellar organization of the aortic media was disrupted in the regions around the coronary artery orifices. The regions around the orifices also exhibited higher cell density than other areas of the aortic media, and mitotic figures were frequently observed within the vicinity of coronary artery orifices. Non-blood cells and cellular fragments were observed within the coronary orifice. Some of the endothelial cells were irregularly shaped, exhibited few junctional contacts with neighboring endothelial cells, and, in some instances, actually protruded into the lumen of the coronary artery orifice.

Coronary Orifice Remodeling and Arterial Development

Light microscopy.

In HH 34–35 hearts, the myocardial cuff had assumed a more ringlike appearance (Fig. 3a). Very well developed aortic valve leaflets, dilated aortic sinuses, and a thickened aortic media were also observed. Serial sections revealed definitive left and right coronary arteries 30–40 μm in diameter, that were continuous with coronary artery orifices in the aortic sinuses. Only one arterial orifice could be seen in each of the left or right aortic sinuses. The walls of the coronary vessels seemed thicker than in previous stages, suggesting development of the tunica media and adventitia. It was also noted that the aortic tissue immediately surrounding the coronary artery displayed a higher cell density and a different histological appearance as compared to other regions of the aorta. As with the younger embryos, no coronary arteries were found attached to either the pulmonary artery or the posterior aortic sinus.

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Figure 3. Coronary artery development and orifice remodeling. A: Routine H&E stained section of an HH 35 heart showing the aorta (AO), leaflets (VL) of the aortic semilunar valve, and the aortic sinuses (AS). A well-developed coronary artery (black arrows) can be seen attached to the left aortic sinus. The periarterial tissue (PA) surrounding the coronary artery displays a higher cell density and a different histological appearance from the tunica media (TM) of the aorta (×150). B: TUNEL stained sections of an HH 35 heart showing aorta (AO), leaflets (VL) of the aortic semilunar valve, and the left aortic sinus (AS). TUNEL positive cells (white arrows) can be seen on either side of the coronary artery orifice and along the margins of the coronary artery. TUNEL positive cells are also observed within the myocardial cuff (MC). (×150) C: TEM micrograph of an HH 35 heart showing the left aortic sinus (AS) and coronary artery (CA). A single endothelial cell (*) is found within the lumen of the coronary orifice. The upper margin of the coronary artery displays a layer of cells (VSMC) in direct contact with the endothelium of the coronary artery. Examination of these cells at higher magnification (data not shown) revealed morphological characteristics consistent with vascular smooth muscle cells of a newly-acquired tunica media (×700). D: Higher magnification of another HH 35 heart showing an endothelial cell (En) protruding into the lumen and characteristics of apoptosis (Apo) in non-blood cells within the lumen of the coronary orifice (CO) (×3,900).

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TUNEL staining.

TUNEL staining of aortas in HH 34–35 embryos displayed a similar pattern to HH 32–33 embryos (Fig. 3b). The number of positive cells in the myocardial cuff was about the same as in HH 32–33 embryos. Positive cells were occasionally observed within the valve leaflets and interspersed in the aortic media and endothelium. There were still, however, numerous TUNEL positive cells associated with the peritruncal capillary plexus, along the proximal portions of the coronary arteries, and adjacent to the developing coronary artery orifices.

Transmission electron microscopy.

In the aortas of HH 34–35 embryos examined with the TEM, irregular non-blood cells and cellular fragments were observed within the lumens of coronary orifices, as were irregularly shaped endothelial cells protruding into the lumens of the coronary orifices (Fig. 3c,d). Cell density within the entire aortic media and along the coronary artery wall had increased relative to earlier stages. Spindle-shaped cells were found approximating the endothelium in the proximal portions of the coronary vessels, suggesting development of a tunica media in a centrifugal (proximal to distal) manner as previously described by Hood and Rosenquist (1992).

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. 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.

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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.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED

The authors wish to recognize Glenn Hoskins (Department of Anatomy, University of Mississippi Medical Center) whose technical expertise and assistance in transmission electron microscopy greatly contributed to this study.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. LITERATURE CITED