Here we identify a novel function for the transcription factor GATA4 as a regulator of angiogenesis in the heart. Our first indication that GATA4 might be a proangiogenic factor came from studies in GATA4-overexpressing transgenic mice. While overexpression approaches are sometimes criticized as being unphysiologic, our inducible strategy bypassed any developmental effect that may have occurred in embryos or neonates, and even in adulthood expression was only 4.6- or 2.5-fold above endogenous GATA4 levels. Indeed, this relatively mild level of overexpression in the adult heart likely models the endogenous increase in GATA4 activity that normally occurs following increased hemodynamic load (Figure A; refs. 23
Microvessel expansion has been reported during pressure overload myocardial hypertrophy in multiple species, including humans (9
), suggesting the possibility that GATA4 overexpression had a primary effect on the hypertrophic response that merely impacted vascular density as a secondary consequence. However, we believe this to be unlikely, since line 21.2 GATA4 transgenic mice (low expressing) showed no cardiac hypertrophy well past 7 months of age, yet they maintained greater capillary density and had enhanced coronary flow. Another interesting feature of the GATA4-driven angiogenic response was its maintenance over long periods of time (at least 6 months), in contrast to cardiomyocyte Akt activation in the heart, which only induced a transient angiogenic response (10
). Likewise, pressure overload stimulation also induces a transient increase in capillary densities with TAC stimulation (9
), but long-term hypertrophy, especially in adult humans, has been associated with a decrease in capillary density and cardiac decompensation, myocyte death, and fibrosis (7
Capillary density in the heart is also tightly coupled to cardiomyocyte growth during development (14
). This program is likely recapitulated in the adult heart that is subjected to injury or physiologic stimulation, given that the hypertrophied myocardium also increases vessel growth to ensure adequate perfusion as the myocyte compartment expands, and inhibition of this “matching” effect in microvessels during hypertrophy was recently shown to promote decompensation (9
). In GATA4-transgenic hearts this increase in capillary density and microvessels resulted in a greater coronary flow reserve and enhanced contractility (+dP
) upon vasodilation in an isolated working heart preparation. This phenomenon is of significant functional importance, since increased myocardial oxygen demand, which is present during increased hemodynamic load or exercise, is mainly satisfied by vasodilation and increased perfusion rather than increased oxygen extraction from the blood (40
). These data imply that GATA4 serves a compensatory and protective role in the heart by inducing the angiogenic program. These results also suggest that activation of GATA4 in the hypertrophic or pressure-loaded heart might serve as an important mechanism for reemploying the developmental angiogenic response in the heart. Indeed, heart-specific Gata4
-deleted mice failed to show enhanced capillary densities following acute pressure overload. More importantly, reinduction of cardiac vascular density in Gata4
-deleted mice using VEGF and angiopoietin-1 adenoviral gene transfer partially rescued ventricular dysfunction following 1 week of pressure overload stimulation. Our results strongly suggest that loss of microvessels from the heart is partially causative in the deterioration observed in Gata4
-deleted mice. Mechanistically, capillary rarefaction can lead to disseminated mild tissue ischemia that likely enhances cell death and functional insufficiency. Although the reduction in capillary density appears somewhat modest in the hearts of Gata4β-Cre
mice, an even lower magnitude change in capillary density was observed in mice with cardiomyocyte-specific deletion of HIF-1α (41
), suggesting that the effect of GATA4 was equal to or stronger than that programmed by HIF-1α. Finally, overexpression of GATA4 in the heart did not induce HIF-1α expression, suggesting that GATA4 can function independent of this angiogenic regulatory factor (data not shown).
In addition to GATA4, the closely related family member GATA6 is expressed in the developing and postnatal heart (42
). Hence, GATA6 might partially compensate for loss of GATA4 in maintaining some degree of capillary density in the heart. Indeed, we recently showed that compound heterozygotes for the traditional Gata4
- and Gata6
-targeted alleles are embryonically lethal with associated defects in the vascular system (43
). Moreover, Ad-GATA6 overexpression in neonatal cardiomyocytes in the HUVEC coculture model produced a similar increase in tube formation and organization compared with Ad-GATA4 (data not shown). These observations collectively suggest that GATA4 and GATA6 proteins function in the regulation of angiogenesis/vasculogenesis, although GATA4 may play a more central role, since it directly responds to diverse pathologic and adaptive stimuli and is phosphorylated by multiple kinases as a means of increasing its activity.
Angiogenesis in the adult heart is regulated by 2 potentially distinct mechanisms (with some partial overlap). The first mechanism is the well-known hypoxia-induced response through HIF-1α, which can directly regulate endothelial cell activity and increase capillaries (19
). Indeed, in the human myocardium, HIF-1α activation was only detected in areas that were directly ischemic (44
), although a second mechanism was suggested by the observation that HIF-1α was activated by mechanical overload in mice (45
). The second mechanism is associated with a general stress response to the heart following acute hemodynamic overload or exercise- or pregnancy-induced hypertrophy (9
). Such a response would allow the myocardium to coordinate an increase in microvessels in synchrony with an ensuing hypertrophy response, but without the induction of tissue ischemia. Moreover, this load-induced angiogenic response would protect the heart from developing ischemia in the first place, possibly delaying decompensation. Here we identify GATA4 as a critical regulator of this load-induced angiogenic response in the heart, although we do not address its interplay with the hypoxia response or potential coordination with HIF-1α.
The angiogenic response mediated by GATA4 appears to initiate an entire program of angiogenic cytokines and growth factors, as revealed by Affymetrix array screening. For example, GATA4 DTG hearts showed upregulation of mRNAs for syndecan-1 (47
), tachykinin-1 (48
), laminin β3 (49
), and more (Table ). Moreover, tissue factor pathway inhibitor (tfpi), a known inhibitor of angiogenesis, was found to be downregulated (50
). A more rigorous quantitative analysis of angiogenic factors by RNase protection assay also revealed significant increases in VEGF and angiopoietin-1 (which were not changed enough to be detected on the Affymetrix array). We believe that induction of this program may be partially direct, since the Vegfa
gene was a binding target for GATA4 and given the reciprocal regulation of angiogenesis that occurred in GATA4 gain- and loss-of-function approaches in culture and in the adult mouse heart. Importantly, pharmacological blockade of the VEGF receptor 2 by CBO-P11 completely inhibited GATA4-induced capillary formation in vitro, indicating that induction of VEGF-A is an essential part of the GATA4-induced angiogenic gene program. Therefore, GATA4 can directly regulate VEGF-A expression and the angiogenic program in the heart through a hypoxia-independent mechanism. These results also highlight a novel mechanism whereby GATA4 promotes cardiac compensation following pathologic stimulation, suggesting a possible novel therapeutic approach to inducing the angiogenic program by GATA4 gene therapy or by other means of increasing its activity.