Correspondence

Angiogenesis Factors in Acute Myocardial Ischemia and Infarction

N Engl J Med 2000; 343:148-149July 13, 2000

Article

To the Editor:

We have three comments about the interesting report by Lee et al. (March 2 issue)1 on the expression of hypoxia-inducible factor 1 (HIF-1) and vascular endothelial growth factor (VEGF) in acute myocardial ischemia and infarction.

First, the analysis of the sections of myocardium stained with hematoxylin and eosin may be suboptimal. According to the legend for Figure 1, the specimen from a patient with acute ischemia (Panel C) has wavy fibers and intact nuclei. The reference that the authors cite for the histopathological diagnosis of acute myocardial ischemia and infarction, however, states that wavy fibers are seen at the edges of acute infarcts and that cytoplasmic alterations in infarction occur before the nuclear changes.2 In Panel C, the wavy fibers appear shrunken, and many of the myocytes seem to be anuclear, as compared with the normal myocardium (Panel D), further supporting the diagnosis of early infarction. Perhaps a more sensitive histologic technique for the detection of myocardial necrosis3 — trichrome staining would probably be the simplest — would help distinguish better between acute ischemia and early infarction. This distinction is important, since the authors conclude that patients with acute ischemia and those with early infarction differ with respect to VEGF messenger RNA and protein expression.

We also question the use of diluted horse serum as a negative control for the monoclonal antibodies to VEGF and HIF-1. Harlow and Lane suggest that the negative control for monoclonal antibodies should be from the same source (supernatant, ascites fluid, or antibody) as the test antibody.4

Finally, Lee et al. propose that the induction of HIF-1 and, consequently, of VEGF results in angiogenesis and vascular remodeling, which may limit the size of the infarct. A plausible, but certainly not contradictory, teleologic explanation is that angiogenesis is an essential component of granulation tissue, part of the healing process.2 VEGF appears to contribute to the formation of granulation tissue in wound healing in humans, at least at extracardiac sites.5

John Wurzel, M.D.
Bruce I. Goldman, M.D.
Temple University Medical School, Philadelphia, PA 19140

5 References

1. 1

   Lee SH, Wolf PL, Escudero R, Deutsch R, Jamieson SW, Thistlethwaite PA. Early expression of angiogenesis factors in acute myocardial ischemia and infarction. N Engl J Med 2000;342:626-633
   Full Text | Web of Science | Medline

2. 2

   Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease. 6th ed. Philadelphia: W.B. Saunders, 1999:558-60.
   

3. 3

   Vargas SO, Sampson BA, Schoen FJ. Pathologic detection of early myocardial infarction: a critical review of the evolution and usefulness of modern techniques. Mod Pathol 1999;12:635-645
   Web of Science | Medline

4. 4

   Harlow E, Lane D. Antibodies: a laboratory manual. Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory, 1988:391.
   

5. 5

   Pokharel RP, Maeda K, Yamamoto T, et al. Expression of vascular endothelial growth factor in exuberant tracheal granulation tissue in children. J Pathol 1999;188:82-86
   CrossRef | Web of Science | Medline

Author/Editor Response

The authors reply:

To the Editor: Our analysis of sections of myocardium stained with hematoxylin and eosin was accurate, not suboptimal. The wavy cardiomyocytes in Panel C of Figure 1 are stretched, buckled, and viable. Hypoxia induces this abnormality within 30 to 60 minutes.1 The cardiomyocytes in Panels A and B are necrotic, with clear-cut hypereosinophilia, coagulation necrosis, vacuolar degeneration (myocytolysis), and nuclear loss. These pathological changes are not present in Panel C. Trichrome staining is not the only technique for making an accurate pathological diagnosis of myocardial ischemia or infarction.

Second, our immunohistochemical methods are valid and well established.2 Our negative control was used to test for false positive results. We determined whether the secondary antibody and color-reaction substrates, rather than the primary antibody, would have immunoreactivity by simply omitting the primary antibody from our negative-control slides and leaving the rest of the steps and reagents exactly the same. The protocol for leaving out the primary antibody and using serum from which the secondary antibody is made as a negative control is widely used in the scientific community.3,4 Indeed, we have been able to verify the presence of the α subunit of HIF-1 and VEGF protein in our myocardial samples, using standard techniques and either mouse monoclonal primary antibodies (with mouse blocking serum as a control) or horse polyclonal primary antibodies (with horse blocking serum as a control).

Third, the major components of granulation tissue are myofibroblasts and vascular endothelial cells, with the formation of small blood vessels.5 The cardiac-biopsy specimens in Panels A, B, and C of Figure 1 show no evidence of granulation tissue with fibroblastic proliferation. The specimen in Panel A represents the onset of infarction less than 24 hours before surgery, Panel B the onset of infarction 24 to 120 hours before surgery, and Panel C ischemia less than 48 hours before surgery. The formation of granulation tissue occurs 7 to 10 days after acute myocardial infarction, which is definitely later than the periods represented by the specimens shown in Figure 1.1

The early expression of angiogenesis factors such as VEGF in our investigation of acute myocardial infarction is due to an increase in HIF-1α. A transcriptional activator of VEGF, HIF-1α initiates early angiogenesis.

Sang H. Lee, M.D.
Paul L. Wolf, M.D.
Patricia A. Thistlethwaite, M.D., Ph.D.
University of California, San Diego, San Diego, CA 92103-8892

5 References

1. 1

   Cotran RS, Kumar V, Collins T, eds. Robbins pathologic basis of disease. 6th ed. Philadelphia: W.B. Saunders, 1999:558.
   

2. 2

   Makinen T, Olofsson B, Karpanen T, et al. Differential binding of vascular endothelial growth factor B splice and proteolytic isoforms to neuropilin-1. J Biol Chem 1999;274:21217-21222
   CrossRef | Web of Science | Medline

3. 3

   An FQ, Matsuda M, Fujii H, Matsumoto Y. Expression of vascular endothelial growth factor in surgical specimens of hepatocellular carcinoma. J Cancer Res Clin Oncol 2000;125:153-160
   CrossRef | Web of Science

4. 4

   Horobin RW. Understanding histochemistry: selection, evaluation, and design of biological stains. New York: John Wiley, 1998:52-3.
   

5. 5

   Bennington JL. Saunders dictionary and encyclopedia of laboratory medicine and technology. Philadelphia: W.B. Saunders, 1984:672.
   

Citing Articles (2)

Citing Articles

1. 1

   Stefan A.M. Paul, Jonathan W. Simons, Nicola J. Mabjeesh. (2004) HIF at the crossroads between ischemia and carcinogenesis. Journal of Cellular Physiology 200:1, 20-30
   CrossRef

2. 2

   Takeshi Hayashi, Nobuo Noshita, Taku Sugawara, Pak H. Chan. (2003) Temporal Profile of Angiogenesis and Expression of Related Genes in the Brain After Ischemia. Journal of Cerebral Blood Flow & Metabolism166-180
   CrossRef