Morphology and Density of Initial Lymphatics in Human Myocardium Determined by Immunohistochemistry

Abstract

Background: Myocardial edema is a common finding in congestive heart failure, transplant rejection, and cardiomyopathy. Although pathological alterations in the lymphatic morphology are part of the underlying disease, knowledge on these changes is limited. However, lymphatic morphology may be investigated by immunohistochemical staining for fms-like tyrosine 4 kinase (flt-4), which is specific for lymphatic endothelium in adult tissue. Methods: We used myocardial tissue of ventricular out flow tract taken from five human semilunar valves harvested as allografts but unsuitable for implantation for analysis, performing immunohistochemical staining for flt-4 with a commercially available antibody. Lymphatic morphometry was completed according Gundersen method. Results: Immunohistochemical staining for flt-4 resulted in successful labeling of lymph capillaries in adult human myocardium. Lymph capillary density was calculated as 50.7 ± 12.5 per mm² and average diameter was 3.7 ± 0.7 µm. Conclusions. Lymph capillary morphology in human myocardium may be successfully determined by immunohistochemical staining for flt-4. Tissue samples as small as myocardial biopsies may be used for analysis. Using this method, morphological changes in myocardial lymphatics may be investigated in various cardiovascular diseases.

Key words

Myocardial edema - lymphatics - flt-4 - allograft

References

• 1 Laine G A. Microvascular changes in the heart during chronic arterial hypertension.  Circulation Research. 1988;  62 953-960
• 2 Davis K L, Laine G A, Geissler H J, Mehlhorn U, Brennan M L, Allen S J. Effects of myocardial edema on the development of myocardial interstitial fibrosis.  Microcirculation. 2000;  7 269-280
• 3 Hosenpud J D, Douglas J N, Cobanoglu M A, Floten H S, Conner R M, Starr A. Serial Echocardiographic Findings Early After Heart Transplantation: Evidence for Reversible Right Ventricular Dysfunction and Myocardial Edema.  Transplant. 1987;  6 343-347
• 4 Marchetti C, Poggi P, Calligaro A, Arbustini E. Lymphatic system in human dilated cardiomyopathy.  J Submicro Cytol Pathol. 1988;  20 701-708
• 5 Feola M, Merklin R, Cho S, Brockman S K. The Terminal Pathway of the Lymphatic System of the Human Heart.  Ann Thorac Surg. 1977;  24 531-536
• 6 Ullal S R, Kluge T H, Kerth W J, Gerbode F. Anatomical studies on lymph drainage of the heart in dogs.  Ann Surg. 1972;  1 (75) 305-310
• 7 Miller A J. The study of the lymphatics of the heart: an overview.  Microcirculation, Endothelium and Lymphatics. 1985;  2 349-360
• 8 Geissler H J, Davis K L, Laine G A, Brennan M L, Mehlhorn U, Allen S J. Contamination of lymph from the major prenodal cardiac lymphatic in dogs.  Am J Physiol. 1999;  276 (5 Pt2) H1795-H1800
• 9 Irrthum A, Karkkainen J, Devriendt K, Alitalo K, Vikkula M. Congenital hereditary lymphedema causes by a mutation that inactivates VEGFR3 tyrosine kinase.  Am J Hum Genet. 2000;  67 295-301
• 10 Kaipainen A, Korhonen J, Mustonen T, Hinsbergh V W van, Fang G H, Dumont D, Breitman M. et al . Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.  Proc Natl Acad Sci. 1995;  92 3566-3570
• 11 Kukk E, Lymboussaki A, Taira S, Kaipainen A, Jeltsch M, Joukov V, Alitalo K. VEGF-C receptor binding and pattern of expression with VEGF-3 suggests a role in lymphatic vascular development.  Development. 1996;  122 3829-3837
• 12 Gundersen H J. Notes of the estimation of the numerical density of arbitrary profiles.  J Microsc. 1977;  1 (11) 219-223
• 13 Okada E. Alteration of epicardial lymphatics in lymphostatic canine hearts.  Heart Vessels. 1988;  4 215-220
• 14 Poggi P, Marchetti C, Calligaro A, Casasco A. Morphometric data on the lymph capillaries of the ventricles of the rabbit heart.  Acta Anat. 1989;  136 185-189
• 15 Boucher Y, Roberge S, Roy P E. Ultrastructural comparative study on lymphatic capillaries of the subendocardium, myocardium, and subepicardium of the heart left ventricle.  Microvasc Res. 1985;  29 305-319
• 16 Banerji S, Ni J, Wang S X, Clasper S, Su J, Tammi R, Jones M, Jackson D G. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.  J Cell Biol. 1999;  144 (4) 789-801
• 17 Prevo R, Banerji S, Ferguson D J, Clasper S, Jackson D G. Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.  J Biol Chem. 2001;  276 (22) 19 420-19 430
• 18 Cursiefen C, Schlotzer-Schrehardt U, Kuchle M, Sorokin L, Breiteneder-Geleff S, Alitalo K, Jackson D. Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.  Invest Ophthalmol Vis Sci. 2002;  43 (7) 2127-2135
• 19 Wilting J, Papoutsi M, Christ B, Nicolaides K H, Kaisenberg C S von, Borges J, Stark G B, Alitalo K, Tomarev S I, Niemeyer C, Rossler J. The transcription factor Proxl is a marker for lymphatic endothelial cells in normal and diseased human tissues.  FASEB J. 2002;  16 (10) 1271-1273
• 20 Kubo H, Cao R, Brakenhielm E, Makinen T, Cao Y, Alitalo K. Blockade of vascular endothelial growth factor receptor-3 signaling inhibits fibroblast growth factor-2-induced lymphangiogenesis in mouse cornea.  Proc Natl Acad Sci USA. 2002;  99 (13) 8868-8873
• 21 Lund G, Morin R L, Olivari M T, Ring W S. Serial Myocardial T2 Relaxation Time Measurements in Normal Subjects and Heart Transplant Recipients.  J Heart Transplant. 1988;  7 274-279