Myocardial Hypertrophy Overrides the Angiogenic Response to Hypoxia

Background: Cyanosis and myocardial hypertrophy frequently occur in combination. Hypoxia or cyanosis can be potent inducers of angiogenesis, regulating the expression of hypoxia-inducible factors (HIF), vascular endothelial growth factors (VEGF), and VEGF receptors (VEGFR-1 and 2); in contrast, pressure overload hypertrophy is often associated with impaired pro-angiogenic signaling and decreased myocardial capillary density. We hypothesized that the physiological pro-angiogenic response to cyanosis in the hypertrophied myocardium is blunted through differential HIF and VEGF-associated signaling. Methods and Results: Newborn rabbits underwent aortic banding and, together with sham-operated littermates, were transferred into a hypoxic chamber (FiO2 = 0.12) at 3 weeks of age. Control banded or sham-operated rabbits were housed in normoxia. Systemic cyanosis was confirmed (hematocrit, arterial oxygen saturation, and serum erythropoietin). Myocardial tissue was assayed for low oxygen concentrations using a pimonidazole adduct. At 4 weeks of age, HIF-1α and HIF-2α protein levels, HIF-1α DNA-binding activity, and expression of VEGFR-1, VEGFR-2, and VEGF were determined in hypoxic and normoxic rabbits. At 6 weeks of age, left-ventricular capillary density was assessed by immunohistochemistry. Under normoxia, capillary density was decreased in the banded rabbits compared to non-banded littermates. As expected, non-hypertrophied hearts responded to hypoxia with increased capillary density; however, banded hypoxic rabbits demonstrated no increase in angiogenesis. This blunted pro-angiogenic response to hypoxia in the hypertrophied myocardium was associated with lower HIF-2α and VEGFR-2 levels and increased HIF-1α activity and VEGFR-1 expression. In contrast, non-hypertrophied hearts responded to hypoxia with increased HIF-2α and VEGFR-2 expression with lower VEGFR-1 expression. Conclusion: The participation of HIF-2α and VEGFR-2 appear to be required for hypoxia-stimulated myocardial angiogenesis. In infant rabbit hearts with pressure overload hypertrophy, this pro-angiogenic response to hypoxia is effectively uncoupled, apparently in part due to altered HIF-mediated signaling and VEGFR subtype expression

Figure 6

<p>A) Representative autoradiograph of electrophoretic shift assay (EMSA) of specific binding of HIF-1α nuclear protein. Increased activity of HIF-1α was detected in hypertrophic myocardium under normoxic conditions with further elevation under hypoxia. As a positive HIF-1α control, nuclear extracts were obtained from COS-7 (COS) cells treated with 0.15 mM CoCl₂ for 16 hours and subjected to parallel EMSAs. B) Densitometry analysis of HIF-1α EMSAs.</p

Cumulative data of blood gas analyses of sham-operated animals that have been exposed to chronic hypoxia.

<p>The shown data demonstrate immediate changes of arterial oxygen saturation upon exposure to hypoxia (FiO₂ = 0.12) (2a) followed by increased hematocrit (2b). The banded animals responded to hypoxia in the same manner when compared to non-banded animals (not shown) (*p<0.01).</p

Summary of serum erythropoietin concentrations in mIU/mL.

<p>Hypoxia increased EPO concentrations equally and significantly in both the sham and banded animals. Data are Mean±S.D., N = 6–8 animals/group.</p

Indirect visualization of hypoxia in the myocardium.

<p>Animals from all four experimental groups were injected with 100 mg/kg pimonidazole intraperitoneally 30 minutes before euthanasia. The microscopic images show the detection of pimonidazole adducts, which form only under hypoxic conditions (pO₂<10 mm Hg at tissue level). The top panel (A–D) shows antibody-based detection of pimonidazole adducts (green fluorescence); the bottom panel (E–H) demonstrates staining of nuclear DNA with DAPI (blue; the scale bar represents 25 µm). Animals that were exposed to chronic hypoxia showed positive staining for pimonidazole adducts, while animals that have been housed under normoxic conditions were tested negative for pimonidazole adducts. All images were acquired with identical acquisition settings (exposure time, light intensity, and neutral density filters). Surprisingly, although no hypoxia was observed in animals of the BAND NORM group, those animals showed increased expression of HIF-1α.</p

Summary of densitometry analyses of normalized protein levels of HIF-1α, VEGFR-1, HIF-2α, VEGFR-2, and VEGF obtained by immunoblotting using lysates isolated from aortic banded and sham operated animals exposed to either normoxia or hypoxia.

<p>Results are based upon 3–5 hearts/group.</p

Capillary density.

<p>A) Representative micrographs of left ventricular cross-sections (6 µm thickness) showing capillary vessels in all groups (as visualized by FITC-labeled lectin, scale bar represents 100 µm). B) Vessel density quantification assessed by computer-based image analysis is shown in 4b (*p<0.01, shown as mean±SE). As a physiologic response to chronic hypoxia, sham operated animals showed a significant increase in capillary density. Animals with left ventricular hypertrophy showed a relative decrease in capillary density under normoxia. Chronic hypoxia failed to induce an increase of capillary density in hypertrophying LV myocardium.</p

Cumulative data of the LV-to-bodyweight ratio of all experimental groups at 4 weeks of age (*p<0.01).

<p>The animals that underwent aortic banding developed significant LV hypertrophy starting at one week after surgery. Although hypoxia affected the overall growth of the animal, the development of LV hypertrophy was not significantly altered.</p