Coronary vascular patterning during embryonic development
To observe the sequence of coronary vascular patterning, endothelial cell formation on the surface of the heart was analyzed in wildtype embryos at different developmental stages from E10.5 to E13.5. Pecam1 (platelet-endothelial cell adhesion molecule 1) (Baldwin et al., 1994
) whole mount staining was performed on isolated embryonic hearts to analyze the nascent endothelial tube formation in vivo
. At E10.5, a few Pecam1-positive cells were observed on the posterior surface of the heart (). At E11.5, primitive, yet unrefined, endothelial network or patches were observed in the atrioventricular junction (). By E12.5, these endothelial tubes extended from the atrioventricular junction inferiorly and laterally to the apical and lateral regions of the ventricles, forming a more elaborate vascular network () that began to encroach upon the anterior surface of the heart (). By E13.5, many endothelial tubes had coalesced to form more distinct vascular pattern in the posterior surface (). Endothelial tubes became apparent at the lateral border of the heart, and a number of endothelial nodules appeared on the anterior surface of the heart (). This sequence of events suggests that cells at the distal end of endothelial tubes proliferate to expand the vascular formation or new endothelial cells are recruited to keep the progression of vascular network. Retroviral cell tagging studies of chick hearts suggest that coronary vessels grow discontinuously by recruiting and coalescing new endothelial cells (Mikawa and Fischman, 1992
Vascular patterning of the coronaries during embryonic development
Calcineurin inhibition leads to abnormal coronary angiogenesis
To study the role of calcineurin-NFAT signaling in coronary vessel development, we used cyclosporine A (CsA) to inhibit calcineurin activity in embryos at different gestational ages. As we described previously, CsA effectively inhibits calcineurin activity in embryos within 3 hours of administering to pregnant mice (Chang et al., 2004
). Its level in embryos drops to the control background level within 24 hours after CsA treatment, providing precision of one developmental day to define the window of calcineurin-NFAT action.
Pregnant mice were treated with CsA (50 mg/kg, intraperitoneal injection, twice a day) starting from E9.5, E10.5 or E11.5, and embryos were harvested at E12.5 for analysis of coronary vasculature patterning. Embryos exposed to CsA in utero
were grossly indistinguishable from untreated embryos at E12.5, regardless of the starting time of CsA treatment (). Hearts from CsA-treated embryos were also similar in size to littermate controls (), suggesting that there was no gross developmental delay at E12.5 with CsA treatment. Coronary vessel development was analyzed by Pecam1 whole mount staining of E12.5 hearts. E12.5 was chosen as the time point for analysis because embryos treated with CsA died at early E13 due to heart valve defects (Chang et al., 2004
), precluding further examination of coronary development at or beyond E13. Pecam1 whole mount analysis showed an extensive network of coronary endothelial cells in untreated embryos at E12.5 whereas embryos exposed to CsA at E10.5 showed limited endothelial patterning (). The endothelial tubes were fused and restricted to the atrioventricular junction, failing to form an elaborate endothelial network. In contrast, embryos exposed to CsA at E9.5–10.5 or E11.5–E12.5 displayed normal coronary endothelial patterning at E12.5 (). Also, embryos exposed to CsA during these windows from E9.5 to E12.5 showed no endothelial patterning abnormalities in the peripheral vascular beds of cranial, intersomitic and dorsal regions of the embryo (data not shown), consistent with previous reports that calcineurin functions at E7.5–E8.5 to regulate peripheral angiogenesis (Graef et al., 2001
). Taken together, these observations indicate a specific temporal requirement of calcineurin at E10.5 for normal coronary vessel development.
Calcineurin-NFAT signaling is not required in epicardial or myocardial cells for embryonic coronary angiogenesis
The next question becomes where the calcineurin-NFAT functions to regulate coronary development. Coronary development requires reciprocal signaling between neighboring cells or tissues that include epicardial, myocardial, vascular smooth muscle cells and endothelial cells. Since calcineurin is widely expressed in all these cells (Chang et al., 2004
), calcineurin could function in any of these cell types to regulate coronary vessel formation. To determine the cellular site of calcineurin action during coronary angiogenesis, we performed tissue-specific gene deletion of the calcineurin regulatory subunit (Cnb1) using the murine cre-lox genetic method.
To study the roles of calcineurin in epicardial or myocardial cells for coronary vascular patterning, we deleted Cnb1
in epicardial and myocardial cells, using Gata5Cre
mouse lines, respectively. The Gata5
promoter in Gata5Cre
mouse line directs the expression of Cre recombinase to epicardial cells (Merki et al., 2005
), which give rise to vascular smooth muscle cells of coronary vessels (Mikawa and Fischman, 1992
; Mikawa and Gourdie, 1996
; Perez-Pomares et al., 2002
). The Gata5Cre
activity is evident by E9.25 in the proepicardial cells, and by E9.5–E10.0 in the epicardial cells (Merki et al., 2005
). Epicardial-restricted Cnb1
mutant mice (Gata5Cre;Cnb1F/F
) were grossly normal () and displayed no defects in endothelial patterning at E12.5 by whole mount Pecam1 staining (). Furthermore, Gata5Cre;Cnb1F/F
mice lived to adulthood, and they were indistinguishable from their wildtype littermates at birth (data not shown). This observation indicates that calcineurin in epicardial or in epicardially-derived cells is not required for coronary vessel formation.
Calcineurin-NFAT signaling is not required in epicardial cells or myocardial cells for coronary vessel patterning
To study the role of calcineurin in the myocardial cells during coronary development, we used SM22αCre
mouse line to delete Cnb1
in myocardial cells (). Cre-recombinase driven by the SM22α
promoter is active in both vascular smooth muscle cells and myocardial cells. The SM22αCre
expression in myocardial cells occurs as early as E9.0 as described previously (Stankunas et al., 2008
; Umans et al., 2007
embryos were grossly normal () and showed normal coronary endothelial patterning at E12.5 (). Sm22αCre;Cnb1F/F
mice also lived to adulthood with no evidence of coronary patterning defects at birth (data not shown). These findings indicate that calcineurin is not essential in myocardial or vascular smooth muscle cells for coronary angiogenesis.
To verify the deletion of Cnb1 from epicardial and myocardial cells, we performed calcineurin immunostaining in Gata5Cre;Cnb1F/F and Sm22αCre;Cnb1F/F embryos. We found that calcineurin was indeed removed from proepicardial cells of Gata5Cre;Cnb1F/F embryos by E9.5 () and from myocardial cells of Sm22αCre;Cnb1F/F embryos by E10.5 () before the critical window when calcineurin was required for coronary angiogenesis (). These observations thus validate the conclusion that calcineurin does not function in epicardial, myocardial or vascular smooth muscle cells to regulate early coronary development.
Endothelial calcineurin-NFAT signaling is essential for coronary vascular patterning
To examine endothelial calcineurin function, we used a Tie2Cre
mouse line to direct the deletion of Cnb1
in endothelial cells (Chang et al., 2004
; Kisanuki et al., 2001
). As we reported previously, this Tie2Cre
activity is detectable in endothelial cells by E8.0, and calcineurin is deleted in endocardial cells by E9.5 (Chang et al., 2004
embryos showed no gross developmental defects compared to their wildtype littermates at E12.5 (). The Pecam1 whole mount staining of Tie2Cre;Cnb1F/F
hearts at E12.5 showed limited endothelial branching in contrast to an extensive network of endothelial tubes of the littermate control (). Coronary endothelial cells in Tie2Cre;Cnb1F/F
hearts were fused and limited to the atrioventricular junction. These cells were unable to extend apically or laterally in the heart (). Histological sections of the wildtype hearts showed that Pecam1-positive endothelial cells assembled to form tubes that extended to the lateral and apical parts of the heart, whereas in the Tie2Cre;Cnb1F/F
hearts Pecam1-positive cells were limited to the basal portion of the ventricles with no endothelial cells reaching the apical region of the heart (). These endothelial patterning defects in Tie2Cre;Cnb1F/F
embryos phenocopy the defects observed in CsA-treated embryos (). Combined with the data obtained from mice lacking Cnb1
in epicardial and myocardial cells, these findings demonstrate that calcineurin is specifically required in endothelial cells to regulate endothelial assembly in early coronary vascular development.
Endothelial Calcineurin-NFAT signaling is required for normal coronary vessel patterning
The next question became whether endothelial calcineurin is also required for peripheral vascular patterning in developing embryos. We performed Pecam1 whole mount immunostaining of embryos at E10.5 and E11.5, and observed no significant differences in the peripheral vasculature between wildtype and Tie2Cre;Cnb1F/F
embryos. Cranial, intersomitic and dorsal vessels of Tie2Cre;Cnb1F/F
mutant embryos appeared normal (). Of note, these Tie2Cre;Cnb1F/F
embryos died at E13 due to heart valve defects, consistent with previous reports () (Chang et al., 2004
). These studies suggest that calcineurin in endothelial cells is not essential for peripheral vascular patterning. The differential effects of endothelial calcineurin in coronary versus peripheral vascular development suggest that distinct mechanisms are involved in the development of these two vascular beds.
We next examined whether NFATc genes were expressed in coronary endothelial cells at E12.5 when they first became easily detectable (). By RNA in situ hybridization, we found that NFATc3 and NFATc4, but not NFATc1 or NFATc2, were expressed in coronary endothelial cells (), suggesting that endothelial NFATc3 and NFATc4 are activated by calcineurin to transduce the signals required for coronary angiogenesis.
To determine the extent of the coronary vasculature defects observed at E12.5 in CsA-treated embryos or in the different genetic models, we measured the percentage of surface area of the ventricles covered by the endothelial network marked by Pecam1 staining (). We found that CsA treatment resulted in a diminished endothelial network compared to that of the control embryos (42.2±5.7 % vs. 100±13.4 %; p<0.01) (). In contrast, coronary endothelial network was not impaired in embryos lacking either epicardial (Gata5Cre;Cnb1F/F) or myocardial (Sm22αCre;Cnb1F/F) Cnb1 (). However, in embryos lacking endothelial Cnb1 (Tie2Cre;Cnb1F/F), the endothelial defects occurred to a similar extent to those caused by CsA treatment (40.6±3.8 %), indicating that the primary effects of CsA treatment on coronary angiogenesis were mediated through endothelial calcineurin.
Quantitation of the coronary endothelial network formation and recruitment of perivascular supporting cells
Next we asked whether the Cnb1
-null endothelial cells were capable of recruiting pericytes to form a vessel wall. We analyzed the expression of a marker of pericytes, Pdgfrb, to assess the assembly of endothelial cells and pericytes in Tie2Cre;Cnb1F/F
embryos. By co-immunostaining of Pecam1 (to mark endothelial cells) and Pdgfrb (to mark pericytes), we observed that pericytes were recruited normally to the remaining coronary endothelial cells of Tie2Cre;Cnb1F/F
embryos by E12.5 (). Similarly, recruitment of pericytes to endothelial cells occurred normally in the peripheral vessels of Tie2Cre;Cnb1F/F
embryos (). Together with previous reports showing that calcineurin-NFATc3/c4 is essential for endothelial-pericyte assembly in the cranial and somatic vasculature (Graef et al., 2001
), our observations indicate that vessel wall formation of the peripheral vasculature is regulated by the non-endothelial or peri-vascular functions of calcineurin-NFAT. This is in contrast to the pericyte recruitment in the coronary vascular beds as the peri-endothelial tissues lacking Cnb1
in smooth muscle cells and myocardium of Sm22αCre;Cnb1F/F
embryos had no effects on pericyte/smooth muscle cell recruitment of coronary vessels (data not shown). Unfortunately, vascular smooth muscle cell recruitment to coronary vessels of Tie2Cre;Cnb1F/F
embryos could not be examined since these cells had not yet been recruited to the endothelial tubes before Tie2Cre;Cnb1F/F
embryos died by early E13 ( and data not shown) (Mikawa and Gourdie, 1996
Calcineurin contributes to the initiation of endothelial cells to undergo tubular formation
To understand the cellular defects underlying the coronary phenotype observed in the Tie2Cre;Cnb1F/F
mutants, we first examined the effects of calcineurin inactivation on the proliferation and cell death of endothelilal cells. By Ki67 staining, we found no difference in the percentage of coronary endothelial cells in cell cycle between wildtype and mutant embryos at E12.5 (Supplemental Fig. 1A, B
). Neither was there significant apoptotic cell death in these embryos as shown by TUNEL staining (Supplemental Fig. 1C, D
). Furthermore, human umbilical venous endothelial cells (HUVECs) treated with CsA did not show any significant difference in the BrdU incorporation rate compared with untreated cells. TUNEL assay showed no significant increase in cell death in HUVEC cells in the presence of CsA. Also, there was no difference in the cell number of HUVECs in the presence of CsA (data not shown). These findings suggest that calcineurin does not inhibit endothelial vascular network formation by reducing proliferation or inducing apoptosis of endothelial cells.
Previous studies suggest that calcineurin is required for endothelial cells to form tubular strutures on matrigel cultures (Hernandez et al., 2001
; Rafiee et al., 2004
). Endothelial cells, when cultured on matrigel, spontaneously aggregate and assemble into multicellular capillary-like tubular structures (Folkman and Haudenschild, 1980
; Grant et al., 1991
), recapitulating many aspects of angiogenesis, including cellular migration, differentiation and metalloproteinase activation. However, it is not known whether there is a specific temporal requirement of calcineurin function for that process similar to what we have observed in coronary angiogenesis in developing embryos.
To determine the temporal requirement for calcineurin function in endothelial tube formation, we used CsA to treat HUVECs on matrigel coated plates, starting at different time points in the presence or absence of additional VEGF-A treatment within a 24-hour observation period. HUVEC cells assembled into tube-like structures on the matrigel (), a process that was increased by 30% in the presence of VEGF-A (). When CsA was administered at the time of plating the cells or within the first 6 hours of HUVEC culture, it significantly inhibited tube formation regardless of the presence of VEGF-A (). CsA treatment inhibited tubular formation by 40% in VEGF-A treated cultures, thus offsetting the additional tubular formation due to VEGF-A treatment. Interestingly, many of the HUVECs exposed to CsA during this period aggregated to form large epithelial sheets on the matrigel (), which were never observed in the control HUVECs (). These epithelial sheets resembled the broad endothelial patches present in embryos treated with CsA () or lacking endothelial calcineurin (). These observations suggest that HUVECs and embryonic coronary endothelial cells share similar cellular responses to CsA or calcineurin inhibition.
Calcineurin-NFAT signaling contributes to the initiation of endothelial tube formation
However, when CsA was added after the first 6 hours of HUVEC culture, CsA had no significant effect on HUVEC endothelial tube formation () within the 24-hour observation period, although there were some residual HUVEC epithelial patches. These findings indicate that calcieurin is required predominantly in the early phase of tubular transformation of HUVECs, consistent with a narrow temporal requirement of calcineurin in coronary angiogenesis (). To further determine if an early, but narrow, window of CsA exposure was sufficient to inhibit endothelial tubular formation, we treated HUVECs with CsA for the first 6 hours in culture, washing off CsA, and then continued the culture for another 18 hours to assess the HUVEC tubular formation. We found that HUVECs pulsed with CsA for the first 6 hours exhibited 30–40% reduction in endothelial tubular formation, and these cells aggregated to form large epithelial sheets (either in the presence or absence of additional VEGF stimulation) (), in contrast to HUVECs treated with CsA between the 6th and 24th hour in culture or HUVECs not exposed to CsA (). The latter two experimetal groups showed no difference in endothelial tubular formation. A quantification of endothelial tubular formation in these experiments is represented on . Furthermore, FK506, another inhibitor of calcineurin activity, yielded similar results (data not shown). The reduction of endothelial tubular formation in HUVECs exposed to CsA in the first 6 hours was not caused by cell detachment from the matrigel since the culture supernatantat collected at the 7th and 24th hour of culture among all three experimental groups showed no difference in the number of detached cells (data not shown). Taken together, these findings indicate that calcineurin functions predominantly within the first 6 hours to transduce VEGF-A signaling and trigger endothelial tubular formation.
Similar temporal and cellular responses of endothelial tubular formation to CsA treatment were observed in two additional endothelial cells: human coronary artery endothelial cells (HCAEC, Cambrex, NJ) and mouse embryonic heart endothelial cells (H5V)(Garlanda et al., 1994
) ( and Supplemental Fig. 2
). Thus, these studies demonstrate a narrow time window in which calcineurin-NFAT signaling is required for VEGF-A to trigger endothelial tube formation, similar to the presence of a defined developmental window when endothelial calcineurin is required for coronary angiogenesis.
Calcineurin inhibition does not impair endothelial cell differentiation
To further test the differentiation of CsA-treated endothelial cells and to determine whether endothelial expression of certain angiogenic factors could be compromised in the presence of CsA, we did a survey and examined the expression of several endothelial differentiation and angiogenic factors in CsA-treated HUVECs and HCAECs by quantiative RT-PCR. Included in our survey were ESM-1 (endothelial specific marker1), a proteoglycan involved in endothelial cell adhesion and integrity (Aitkenhead et al., 2002
); CDH5 (VE-Cadherin), involved in remodeling and maturation of endothelial cells (Carmeliet et al., 1999
; Gory-Faure et al., 1999
; Radice et al., 1997
); ENG (endoglin), an accessory protein for the TGF-B receptor complex that promotes endothelial cell proliferation (Lebrin et al., 2004
; Li et al., 1999
); BMPR2 (bone morphogenetic protein receptor type-2), a receptor for BMPs, members of the TGF-β superfamily of ligands involved in endothelial cell proliferation and angiogenesis (Nakaoka et al., 1997
; Zhao, 2003
); TIE2 (angiopoietin-1 receptor), an endothelial receptor tyrosine kinase involved in angiogenesis (Mustonen and Alitalo, 1995
); FLT1 and KDR, receptors of VEGF-A to mediate angiogenesis (Peters et al., 1993
; Quinn et al., 1993
)(). By quantitative RT-PCR, we observed no significant changes of these factors expressed in CsA-treated endothelial cells, suggesting that calcineurin inhibition does not alter the differentiation of endothelial cells. Furthermore, embryos lacking endothelial Cnb1
) had no significant changes of VEGF-A expression (Supplemental Fig. 3
). Therefore, the blunted response of CsA-treated endothelial cells to VEGF-A was not due to mis-regulation of VEGF or VEGF receptor expression, but due to the absence of calcineurin activity to transduce VEGF-VEGFR signaling.
Models of the spatial and temporal actions of calcineurin-NFAT during cardiovascular development