Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

<div><p>Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the <i>sinus venosus</i> and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and <i>sinus venosus</i>. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.</p></div

Human embryonic heart (48 days), hematoxylin-eosin staining.

<p>Hematoxylin-eosin stained heart in a 48 days embryo. The aortic sinus (1) and cushions (2) are indicated. Primitive coronary arteries emerge (arrows) the coronary sinus and dichotomize within the outer myocardium (OM, inset).</p

Human embryonic heart (56 days), CD 34, desmin and α-SMA immune labeling.

<p>Immune labeling for CD34 (A, detail in B), desmin (C) and α-SMA (D) of the future posterior interventricular groove in a 56 days human embryonic heart, oblique-sagittal cut. Endothelial sprouts guided by tip cells (arrow, A and B) invade the outer myocardium. The subepicardial vessels are embedded in a myoid stroma, α-SMA- and desmin-positive (arrowhead in C and D). Myoid cells (arrows in C and D) contact the endothelial sprouts within the outer myocardium.</p

Human embryonic heart (56 days), CD 34 immune labeling.

<p>Immune labeling with CD34 antibodies of a 56 days human embryonic heart, oblique-sagittal cut. General view (A) with detailed area in (B), indicated by the black connector. The CD34 positive endocardial cells cover the ventricular cavity (*). Endocardial tip cells (white arrows) are identified projecting filopodia within the myocardium. The endocardially-derived endothelial networks advance towards the epicardially-derived endothelial networks. ac: <i>apex cordis</i>; P:pericardium.</p

Human embryonic heart (43 days), CD 34, vimentin, CD105, desmin and CD31 immune labeling.

<p>Immune labeling with CD34 (A) and vimentin (B) antibodies of a 43 days human embryonic heart, oblique-sagittal cut at ventricle level. Corresponding epicardial vascular canals are indicated (white arrrows and white arrowheads). The walls of these canals seem to acquire a CD34-positive phenotype and are vimentin-positive. In (A) an active intramyocardial process of sprouting angiogenesis is detailed (inset), being guided by tip cells (double-headed arrows). CD105 immunolabeling of the ventricular wall (C) identifies filopodia-guided processes (arrows) of endocardial sprouting. Desmin-positive reactions were exclusively found (D) in the dorsal wall of the venous sinus (arrows) and in the atrioventricular ring (arrowheads) (VS: venous sinus; RA: right atrium; RV: right ventricle; avc: atrioventricular cushion). CD31-positive endocardial (arrows) and vascular (arrowhead) endothelia were identified. α-SMA intense labeling of the venous sinus (VS) myocardium (arrow) but not of subepicardium (arrowhead).</p

Human embryonic heart (56 days), CD105 (A, B) and vimentin (C, D) immune labeling.

<p>Intramyocardial endothelial networks result from processes of sprouting angiogenesis (arrows) of the underlying CD105-positive and vimentin-positive endocardium. The connector indicates corresponding areas of the left atrium (LA) wall.</p

Human embryonic heart (48 days), CD 34 and CD 31 immune labeling.

<p>Oblique sagittal cut of a 48 days human embryonic heart. Immune labeling of successive slides with CD34 (A, B) and CD31 (C, D) antibodies (1, 1’.atrioventricular cushions; 2.ventricle; 3.primitive atrium; 4.<i>sinus venosus</i>; 5.coronary sinus; 6. endocardial-derived endothelial network in the ventricular wall); insets depict filopodia-projecting endocardial tip cells of the <i>sinus venosus</i> wall (B) and ventricular wall (D). CD105-positive epicardially-derived endothelial tubes (arrows) and intramyocardial endothelia (arrowheads) are indicated in (E). A continuous desmin-positive reaction is identified (F) in the epicardium (arrowheads), but not intramyocardially. Vimentin labeled (G) both the endocardial endothelia (arrow) and the epicardial stroma (arrowhead). Subepicardial vessels are coated by α-SMA-positive pericytes (H, inset, arrows).</p

Human embryonic heart (43 days), CD 34 immune labeling.

<p>Immune labeling with CD34 antibodies of a 43 days human embryonic heart, oblique-sagittal cut at ventricle level. Endocardial tip cells are indicated (arrows).</p