ARTICLE |
Correspondence to: Emma Tham, Dept. of Molecular Medicine, CMM L8:02, Karolinska Institutet, 171 76 Stockholm, Sweden. E-mail: emma.tham@cmm.ki.se
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Summary |
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Angiogenesis is implicated in a variety of human pathologies and may also play a role in the progression of heart failure. We have studied the expression of members of the vascular endothelial growth factor (VEGF) and the angiopoietin families and their receptors in mice lacking the mitochondrial transcription factor A. These mice lack functional respiratory chain activity in their myocytes and develop dilated cardiomyopathy (DCM) postnatally. We studied the hearts of the knockout mice by in situ hybridization, Western blotting analysis, and immunohistochemistry. VEGF-A mRNA and protein levels were elevated in the myocardium of the knockouts. Levels of the hypoxia inducible transcription factor 1 alpha (HIF1) and of glyceraldehyde-3-phosphate dehydrogenase transcripts were also increased, whereas those of angiopoietin-1 and -2 were reduced. Despite the striking upregulation of VEGF-A, there was no increase in capillary density in the knockout hearts. This study suggests that a disturbance in angiogenesis may contribute to the pathogenesis of DCM. (J Histochem Cytochem 50:935944, 2002)
Key Words:
dilated cardiomyopathy, mitochondria, in situ hybridization, VEGF, HIF1, angiopoietin, angiogenesis
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Introduction |
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DILATED CARDIOMYOPATHY (DCM) is a disease of the heart muscle characterized by systolic dysfunction and ventricular dilatation. Heart failure and cardiac arrhythmias develop and are the major causes of death in DCM patients. The etiology of generalized DCM is diverse and includes mutations in sarcomeric genes or mitochondrial DNA, viral infections, toxins, and endocrine disorders (
Angiogenesis is implicated in a variety of human pathologies such as cancer, ICM, and retinopathy (
Mitochondrial dysfunction may be both a cause and a consequence of DCM. Mutations in mtDNA can lead to DCM, often as part of a multiorgan syndrome (
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Materials and Methods |
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Tissue Preparation
Hearts from nine (TfamloxP/TfamloxP + Ckmm-cre) mice at a terminal stage of heart failure (3 weeks of age) and from their healthy (TfamloxP/TfamloxP) littermates were snap-frozen in liquid nitrogen directly after sacrifice (
In Situ Hybridization
Oligonucleotide ISH was performed as previously described (
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The probes were labeled at the 3' end with [35S]-deoxyadenosine-[thio]triphosphate (NEN; Boston, MA) using terminal deoxynucleotidyl transferase (FPLC pure; Amersham Pharmacia Biotech, Uppsala, Sweden) to a specific activity of 1.64 x 109 cpm/µg. Unincorporated labeled nucleotide was removed with QIAquick Nucleotide Removal Kit (Quiagen; Hilden, Germany). Hybridization was performed at 42C for 1618 hr on slides pretreated with 0.005% acetic anhydride in acetone. The hybridization mixture contained 50% formamide (GT Baker Chemicals; Amsterdam, The Netherlands), 4 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate), 1 x Denhardt's solution [0.02% each of polyvinyl-pyrrolidone, bovine serum albumin (BSA), and Ficoll], 1% sarcosyl (N-lauroylsarcosine; Sigma, St Louis, MO), 0.02 M phosphate buffer (pH 7.0), 10% dextran sulfate (Amersham Pharmacia Biotech), 250 µg/ml yeast tRNA (Sigma), 500 µg/ml sheared and heat-denatured salmon sperm DNA (Sigma), and 200 mM dithiothreitol (DTT; Amersham Pharmacia Biotech). After hybridization the slides were washed in 1 x SSC at 60C, dehydrated in ethanol, and exposed to autoradiographic film (Hyperfilm Beta-max; Amersham Pharmacia Biotech) followed by dipping in NTB 2 nuclear track emulsion (Kodak; Rochester, NY). After exposure for 1 day (18srRNA), 5 days (GAPDH, COX, ANF), 4 weeks (VEGF-A), or 5 weeks (all the others), the sections were developed in D-19 developer (Kodak-Pathé; Chalon-Sur-Saone, France) for 4 min, fixed in Unifix (Kodak-Pathé), and mounted. The digitalized darkfield image of the dipped slides of four pairs of hearts was recorded by a Kappa video camera connected to a Leica DM RBE microscope (Mikroskop System; Näsviken, Sweden) and the silver grain density was measured using the NIH Image 1.55VDM program (NIH; Bethesda, MD). The pixel intensity measurements of the silver grains in the tissue have previously been demonstrated to correlate well to the actual number of silver grains (
Western Blotting Analysis
Protein was extracted from the hearts of five terminal stage knockout mice and their littermate controls as well as from unrelated normal mouse heart according to standard procedures. A total of 30 µg of protein and 3 µl kaleidoscope protein size standards (BioRad Laboratories; Hercules, CA) in 1 x SDS buffer containing 50 mM DTT was heated to 85C for 5 min and then electrophoresed on 12% SDS-PAGE gels in a mini-Protean 2 electrophoresis system (BioRad). Proteins were blotted onto 0.45-µm nitrocellulose membranes (Protran; Schleicher & Schuell, Dassel, Germany), stained with Ponceau S, and scanned into the computer to use as a loading control. After blocking in 4% non-fat milk, the membranes were incubated with anti-VEGF-A (sc-7269 or sc-152) 0.4 µg/ml (Santa Cruz Biotechnology; Santa Cruz, CA) or with anti-VEGF-B (AF751) 0.2 µg/ml (R&D Systems; Oxford, UK) at 4C overnight. The membranes were then incubated with secondary horseradish peroxidase-conjugated antibodies at 0.2 µg/ml dilution (Santa Cruz Biotechnology). Detection was carried out by Enhanced ChemiLuminescence (Amersham Pharmacia Biotech) and the proteins were visualized in a CCD camera using the luminescent image analyzer LAS-1000plus (Fujifilm; Tokyo, Japan). The immunostaining pattern of the two VEGF-A antibodies was identical. The ECL signal of antibody sc-7269 and the intensity of the scanned image of the Ponceau S stain (loading control) were quantified using the Image Gauge V3.3 program (Fujifilm). The ratio of the immuno- or Ponceau S staining signal in the knockouts relative to the controls was calculated for each of the five littermate pairs. Recombinant VEGF-B-167 and VEGF-B-186 were produced in COS cells (unpublished data) and used as positive controls, as was a 46-kD fusion protein of VEGF-A (Santa Cruz Biotechnology).
Immunohistochemistry
For blood vessel analysis, immunohistochemistry (IHC) was performed on the same hearts as the ISH experiments. The sections were cut from the third of the heart closest to the apex, with the left ventricle lumen still visible. For the blood vessel staining, 8-µm cryostat cross-sections were thawed onto Superfrost Plus slides (Menzel-Glaser) and stored at -20C until used. The tissues were fixed in 100% ice-cold acetone for 10 min followed by 4% paraformaldehyde for 1 min. After blocking in 3% donkey serum for 30 min, the slides were incubated with primary antibodies in PBSTriton 0.1% buffer overnight at 4C, followed by Cy3-conjugated secondary antibodies at 2.4 µg/ml (Jackson ImmunoResearch Lab; West Grove, PA) for 1 hr at room temperature. Antibodies used were rat anti-mouse CD31/PECAM (01951A from PharMingen; San Diego, CA) at 0.5 µg/ml or rabbit anti-mouse von Willebrand Factor (A0082 from Dako; Glostrup, Denmark) at 16 µg/ml. As negative controls, we omitted the primary antibody or used normal rat/rabbit serum (Dako) at a concentration of 10 µg/ml. The image of the fluorescent blood vessels was recorded by a digital camera (Axiocam; Carl Zeiss) connected to a Zeiss Axioscope 2 MOT microscope. Blood vessels were counted in four pairs of hearts in a total cross-sectional area of 0.56 mm2 per heart. All free-standing positive signals that were not visible branches of another vessel were counted. Vessels crossing the lower and left borders of the field were not counted.
For the VEGF-A staining, 10-µm sections were cut, air-dried for 510 min, and then fixed in 100% ice-cold acetone for 10 min. The slides were blocked with 0.3% H2O2, then with 10% donkey serum in PBS0.1% Tween, followed by blocking endogenous avidin and biotin (avidinbiotin blocking kit SP-2001 from Vector Laboratories; Burlingame, CA). Slides were incubated with rabbit-anti-VEGF-A (sc-152 from Santa Cruz Biotechnology) at 0.2 µg/ml or with normal rabbit serum at 0.4 µg/ml overnight at 4C. After incubation with a donkey anti-rabbit biotinylated secondary antibody (Jackson ImmunoResearch Lab), the slides were incubated with ABC Elite (Vector Laboratories). Antibody detection was performed using the peroxidase substrate diaminobenzidine tetrahydrochloride (DAB) (SK-4100; Vector Laboratories). The sections were counterstained with hematoxylin (Apoteket; Malmö, Sweden).
Statistics
All data are presented as the mean ± SD. Statistical analyses were carried out using GraphPad Prism 3.0 (GraphPad Software; San Diego, CA). The ISH values and the blood vessel counts were analyzed with the paired Student's t-test. The values of the Western blots of VEGF-A and VEGF-B were analyzed using the Wilcoxon signed rank test on the ratios of the knockout to the controls. The results were considered significant at a value of p<0.05.
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Results |
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Further Characterization of the Mouse Model
The knockout mice at a terminal stage of heart failure have been well characterized regarding morphology, histology, and function in previous publications (
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Expression Analysis of VEGF-A and VEGF-B
The most striking alteration among the angiogenesis factors (Fig 1) was the increase in transcript levels of VEGF-A. ISH demonstrated a significant increase in VEGF-A mRNA by 1.7-fold in the DCM hearts of this model. The hybridization signal for VEGF-A displayed a punctate pattern with dense grain accumulations over cells identified as cardiomyocytes by histopathological analysis (Fig 2). However, as previously described (
Western blotting analysis confirmed that VEGF-A was elevated at the protein level. Quantification of the Western blotting signal demonstrated an augmentation of VEGF-A protein in four of the five knockout hearts tested. In contrast, the protein levels of VEGF-B remained constant. Only the 167 amino acid form of VEGF-B was expressed in these mouse hearts (Fig 3).
We performed IHC using an antibody against VEGF-A (A-20) that has previously been used successfully in rat tissues (
ISH of Other Angiogenesis Factors
The transcript levels of the main receptor involved in angiogenesis, VEGFR2, were constant, whereas VEGFR1 was subtly (1.4-fold) elevated in the knockout hearts. VEGF-C had unaltered mRNA signals. Both angiopoietins, their receptor Tie-2, and the orphan receptor Tie-1 displayed a weak mRNA signal over the blood vessels, with a slight reduction in the DCM hearts (Fig 1). Because hypoxia is a powerful inducer of angiogenesis and may involve mitochondrial function, we also analyzed the mRNA of the hypoxia-inducible transcription factor 1 alpha (HIF1), which was upregulated by 2.6-fold in the cardiomyocytes (Fig 1 and Fig 5).
Blood Vessel Density
Because the levels of VEGF-A were elevated, we investigated whether there were any signs of neovascularization in the DCM hearts. Blood vessels (large vessels as well as capillaries) were identified with immunofluorescence staining for CD31/PECAM, a membrane protein expressed constitutively on endothelial cells (
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Discussion |
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In this study we have demonstrated that the expression levels of several of the major factors involved in angiogenesis are affected in DCM of mitochondrial etiology. We examined the expression of the members of the VEGF and angiopoietin families and their receptors by ISH in a mouse model of dilated DCM caused by mitochondrial dysfunction (
Our results diverge from those of
Why does the 3.4-fold increase of VEGF-A fail to elicit an angiogenic response in our mouse model? Angiogenesis is a tightly regulated process and VEGF-A is, for example, only angiogenic in a permissive environment. Exogenous VEGF-A injected into chick embryos failed to induce vascularization of the normally avascular cornea and retina, despite expression of VEGF-A and striking vessel growth in the surrounding tissues (
The presence of leukocytes may determine whether a vascular response will ensue. However, inflammation is not a prerequisite for angiogenesis (
Many other factors may be involved in the regulation of neovascularization. One such factor is the presence or absence of auxiliary proteins, such as the angiopoietins. The angiopoietins act synergistically with VEGF-A (
Angiogenesis has been most extensively studied in ischemic settings. Hypoxia is a powerful inducer of angiogenesis via the action of HIF1 (
mRNA was elevated in our DCM model and has previously been found to rise parallel to progression of heart failure in CHF146 cardiomyopathic hamsters and in post-infarction heart failure in rats (
is also regulated at the protein level, the fact that four of its target genes were stimulated (VEGF-A, VEGFR1, GAPDH, and Glut-1) (
mRNA levels in our model correlate with increased HIF1
activity.
In summary, mice lacking mitochondrial function in their cardiomyocytes develop DCM. They have an increase in VEGF-A mRNA and protein (but not VEGF-B or VEGF-C) in their cardiomyocytes without a concomitant increase in capillary density. In addition, we found an elevation of HIF1 mRNA in the myocardium. In conclusion, upregulation of HIF1
and VEGF-A was not sufficient to induce neovascularization in this model. Therefore, the regulation of angiogenesis appears to be more complex than expected, and this may have implications for the use of angiogenic therapy in patients with, e.g., myocardial infarction.
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Acknowledgments |
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Supported by grants from the Swedish Cancer Society, Axel and Signe Lagerman's Foundation, Sigurd and Elsa Golje's Memorial Fund, the Swedish Medical Society, and Förenade Liv Group Insurance company.
We are grateful to Prof Magnus Nordenskjöld for his support. We thank Dr Nils-Göran Larsson for valuable help and for providing the Tfam knockout mice. We also thank Dr Anders Oldfors (Sahlgrenska Hospital) for expert histopathological guidance.
Received for publication June 8, 2001; accepted January 16, 2002.
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