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Persistent induction of HIF-1 and -2 in cardiomyocytes and stromal cells of ischemic myocardium

JAN STEFFEN JÜRGENSEN^*, CHRISTIAN ROSENBERGER^*, MICHAEL S. WIESENER^*, CHRISTINA WARNECKE^*, JAN H. HÖRSTRUP^*, MICHAEL GRÄFE, SEBASTIAN PHILIPP, WANJA GRIETHE^*, PATRICK H. MAXWELL^||, ULRICH FREI^*, SEBASTIAN BACHMANN, ROLAND WILLENBROCK and KAI-UWE ECKARDT^*,1

^* Department of Nephrology and Medical Intensive Care, Campus Virchow Klinikum
Department of Cardiology, Campus Buch and
Institute of Anatomy, Charité, Humboldt University;
Department of Cardiology, German Heart Center, Berlin, Germany; and the
^|| Imperial College, Renal Section, Hammersmith Campus, London, UK

¹ Correspondence: Department of Nephrology and Medical Intensive Care, Charité, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin. E-mail: kai-uwe.eckardt{at}charite.de

SPECIFIC AIMS

In this study we aimed to investigate the temporo-spatial pattern of induction of the oxygen-regulated -subunits of two principal mediators of adaptation to critically low oxygen levels, hypoxia-inducible transcription factors (HIF) -1 and -2, after myocardial infarction compared with systemic hypoxia. We characterized distinct cell types expressing any HIF- subunit after infarction. Analysis of target genes, HIF RNA changes, and expression in isolated human coronary microvascular endothelial cells complemented the investigation.

PRINCIPAL FINDINGS

1. Neither hypoxia nor myocardial infarction (ischemia) leads to a major increase in HIF-1 and HIF-2 mRNA levels
Under normoxic baseline conditions, HIF-1 and -2 mRNA were readily detectable by RNase protection in adult heart extracts. Exposure of rats to 8% O₂ for 6 h led to a moderate increase of HIF-1 (1.9-fold) and -2 (1.6-fold) mRNA levels. After left coronary artery ligation, HIF-mRNA levels in specimens adjacent to the infarct zone showed a minor increase from 1.14- to 1.6-fold, the highest level for HIF-1 mRNA observed 1 wk post myocardial infarction. A slight increase in HIF-2 mRNA was noted as early as 6 h after myocardial infarction and persisted for 7 days.

2. HIF-1 and HIF-2 protein accumulates after myocardial infarction
Immunohistochemistry failed to detect HIF-1 and HIF-2 protein throughout the myocardium under normoxic conditions. In contrast to the minor increase in HIF mRNA, a pronounced induction of both HIF subunits occurred at the protein level and was seen in cardiomyocytes and stromal cells after myocardial infarction. The intensity of accumulation was comparable to that seen after systemic hypoxia, but the distribution was completely different.

3. The spatial pattern of induction differed for both HIF subunits
Induction of both HIF subunits was already prominent after 6 h and occurred primarily around the infarct. At the anterior and posterior infarct borders, HIF was induced throughout the ventricular wall. HIF protein was consistently observed in subepi- and subendocardial tissue layers, which appeared viable on trichrome staining. After coronary ligation, the peri-infarctious expression persisted in myocardium adjacent to and intermingled with granulomatous and fibrous tissue. Initially, the regional distribution of HIF-1 and -2 signals was similar (Fig. 1 a, c), but from day 1 onward, additional induction of HIF-2 was detected remote from the infarct (Fig. 1b, d ). A thin subendocardial band of positive cells first appeared on the left side of the interventricular septum, which subsequently broadened and, after 4 wk, resulted in homogeneous staining of the septum, noninfarcted parts of the left posterior wall, and less intense scattered staining of the right ventricle.

View larger version (95K): [in this window] [in a new window]   Figure 1. Regional distribution of HIF-1 and HIF-2 after myocardial infarction. Low-power views of HIF-1 (a, b) and HIF-2 (c, d) immunohistochemisry signals were composed after electronic signal amplification and pseudostained in red to demonstrate regional distribution and spatial relationship to the infarct zone after 6 h (a, c) and 7 days (b, d). Induction of both subunits is already prominent after 6 h and occurs primarily around the infarct. HIF protein was consistently observed in or next to viable subepi- and subendocardial tissue layers. After 7 days HIF-2 is also up-regulated in areas distant from the infarct (d).

4. Distinct cell populations express HIF with an overall preponderance of HIF-2 and endothelial cells
Overall, the proportion of cells staining for HIF-2 was significantly higher than for HIF-1. The proportion of positive interstitial cells exceeded the number of positive myocyte nuclei. Especially in capillaries and larger vessels, endothelial cells stained for HIF-2 more frequently than for HIF-1. Some cardiomyocytes and endothelial cells were found to simultaneously accumulate both HIF subunits.

5. Hypoxia induces HIF in isolated human cardiac endothelial cells
To confirm endothelial induction of HIF with an independent technique and further address the hypoxic response of human cardiac endothelium, we isolated microvascular endothelial cells from human hearts and exposed them to low oxygen in vitro. Consistent with the in vivo findings, hypoxic exposure resulted in a marked, time-dependent up-regulation of both HIF-1 and HIF-2 proteins.

6. Infiltrating macrophages express HIF-1 and HIF-2
Double-labeling immunohistochemistry for the macrophage marker ED-1 and HIF-1 or HIF-2 revealed particularly dense staining for HIF in areas with inflammatory infiltrates. HIF induction in macrophages occurred predominantly in cells located in the direct vicinity of necrotic tissue (Fig. 2 ).

View larger version (60K): [in this window] [in a new window]   Figure 2. HIF-1 and HIF-2 expression in infiltrating macrophages. Double-labeling immunohistochemistry for the macrophage marker ED-1 (green fluorescence signal), HIF-1 (red fluorescence, upper panels), and HIF-2 (red fluorescence, lower panels). Overlap of red and green fluorescence signals results in yellow staining of macrophages expressing HIF-1 or HIF-2.

7. Target genes GLUT-1 AND HO-1 are up-regulated in HIF-expressing cells surrounding the infarct
To further examine the functional relevance of regional induction of HIF, immunohistochemistry was performed for hemeoxygenase-1 (HO-1) and glucose transporter 1 (GLUT-1), two genes known to confer different cytoprotective functions under ischemic conditions and to be inducible by HIF-1 in vitro. A highly congruent spatial distribution of nuclear HIF-1 expression and cytosolic expression of GLUT-1 and HO-1 along the border of tissue necrosis was found.

CONCLUSIONS AND SIGNIFICANCE

This study identifies a marked potential for a transcriptional response mediated by the two oxygen-regulated transcription factors, HIF-1 and HIF-2, in different cell types of the heart and indicates widespread relevance of the HIF system in myocardial adaptation to local and systemic hypoxia.

In agreement with the established post-translational regulation of both HIF subunits through the ubiquitin-proteasome pathway under normoxic conditions HIF induction in the heart was seen primarily at the protein level. Different ways of oxygen-independent up-regulation of HIF have been described. The PI3K/Akt pathway mediates HIF-1 expression in nonhypoxic cardiomyocytes exposed to mechanical stress after acute myocardial infarction. The tissue distribution of HIF-1 observed in this study (Fig. 1a , 1b ), however, is consistent with a dominant and sustained role of oxygen tension in its regulation. Within presumably hypoxic areas, HIF-1 induction occurred in cardiomyocytes and stromal cells, including the microvasculature and infiltrating cells. The frequency of HIF-1 induction, however, varied between cell types and was higher in interstitial cells than in cardiomyocytes in their direct vicinity, suggesting that oxygen-sensing mechanisms in nonmyocytes are more sensitive.

Induction of HIF-2 and HIF-1 overlapped (Fig. 1a, c ). Moreover, the hypoxic induction of HIF-1 and HIF-2 in isolated cardiac microvascular endothelial cells was quite similar. However, a comparison of signal distributions of HIF-1 and HIF-2 also revealed two significant differences in vivo. First, both micro- and macrovascular endothelial cells more frequently expressed HIF-2 than HIF-1. This is in keeping with the assumption that HIF-2 plays an important role in hypoxic adaptation of the endothelium postulated when it was first identified, and termed "endothelial PAS" protein. The second potentially important difference is the widespread induction of HIF-2 in myocardial tissue remote from the infarcted areas, which was progressive (Fig. 1d ) and contrasted markedly with the restriction of HIF-1 accumulation to peri-infarctious tissue. Compensatory hypertrophy, higher oxygen consumption, and increased diffusion distances between capillaries and hypertrophied myofibers could all contribute to hypoxia. However, selective induction of the HIF-2 subunit in these areas could indicate that mechanical stress or growth-stimulating factors in cardiac remodeling play an important role in modulating the HIF-2 response, and vice versa.

Additional genes activated by HIF play important roles in angiogenesis. VEGF and the endothelial angiopoietin receptor Tie2 are up-regulated at the border zone of infarcts, consistent with the strong induction of HIF-1 and HIF-2 as demonstrated in the present study (Fig. 1) . Angiogenesis is also dependent in part on cytokines and growth factors produced by activated macrophages that "tunnelize" ischemic regions and form a network. In turn, HIF was recently shown to be essential for activation and infiltration of macrophages and it plays a central role in their glycolytic energy production. Expression of HIF-1 and HIF-2 in macrophages at the border of infarcted myocardium is in tune with these findings and illustrates these critical processes (Fig. 2) .

HIF proteins are attractive targets for therapeutic interventions aimed at improving myocardial oxygen supply. Inhibition of ubiquitin-proteasome-dependent degradation of HIF-1 was found to enhance myocardial vascularity. Exogenous application of DNA encoding an HIF-1/VP 16 hybrid transcription factor enhanced angiogenesis and reduced infarct size in another rodent myocardial infarction model. The pattern of HIF activation as defined in this study may help to tailor therapeutic and perhaps isoform-specific interventions to complement the endogenous response (Fig. 3 ).

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram of cardiac HIF induction. LV, left ventricle; orange, vital myocardium; yellow, infarction; brown, HIF-1 and -2.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1605fje; doi: 10.1096/fj.04-1605fje

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