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J Thorac Cardiovasc Surg 2000;120:707-711
© 2000 The American Association for Thoracic Surgery

Cardiopulmonary Support and Physiology

Exogenous hyaluronidase induces release of nitric oxide from the coronary endothelium

Paulo R. B. Evora, MD, PhD, Paul J. Pearson, MD, PhD, Yeow Leng Chua, MD, Berent Discigil, MD, Hartzell V. Schaff, MD

From the Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minn.

Supported in part by The FAPESP-Fundacao de Amparo a Pesquisa do Estado de São Paulo and the Mayo Foundation.

Address for reprints: Paulo R. B. Evora, MD, PhD, Rua Rui Barbosa, 367, 14015-120 Ribeirao Preto, São Paulo, Brazil (E-mail: prbevora{at}keynet.com.br).


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective: Hyaluronidase, an endogenous enzyme that hydrolyzes mucopolysaccharides, has been shown to enhance myocardial protection when added to preservation solutions. In addition, hyaluronidase infusion reduces injury to ischemic myocardium. Endothelium-derived nitric oxide is an endogenous vasodilator that prevents leukocyte adhesion to the intima and inhibits platelet adhesion and aggregation in the coronary artery. Experiments were undertaken to determine whether the protective action of hyaluronidase could be mediated by the endogenous release of nitric oxide.
Methods: Segments of coronary artery, with and without endothelium, were placed in organ chambers (25 mL) to measure isometric force. Blood vessel segments were contracted with prostaglandin F2{alpha} (2 x 10–6 mol/L) and exposed to hyaluronidase (3-15 units).
Results: Hyaluronidase induced vasodilation of arteries with intact endothelium but not of arteries without endothelium (n = 6, P < .05). Endothelium-dependent vasodilation to hyaluronidase was blocked by NG-monomethyl-L-arginine (10–5 mol/L), an inhibitor of nitric oxide synthesis from L-arginine (n = 6, P < .05). Inhibition of vasodilation by NG-monomethyl-L-arginine was reversed by L-arginine (10–4 mol/L) but not D-arginine (10–4 mol/L; n = 6, each group). Vasodilation to hyaluronidase also was inhibited by hemoglobin (2 x 10–6 mol/L), a scavenger of the nitric oxide radical (n = 6, P < .05).
Conclusions: Hyaluronidase induces the release of nitric oxide from the coronary endothelium. Because nitric oxide, an endogenous vasodilator, inhibits leukocyte adhesion to the intima in addition to inhibiting platelet adhesion and aggregation, stimulated production of endothelium-derived nitric oxide by exogenous hyaluronidase could be the mechanism of the protective action of hyaluronidase infusion.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Hyaluronidase modifies the permeability of connective tissue through the hydrolysis of hyaluronic acid, a polysaccharide found in the intercellular ground substance. In animal and in human studies, hyaluronidase infusion has been shown to reduce ischemic injury due to coronary occlusion.Go Go 1-4 In addition, myocardial preservation solutions containing hyaluronidase have been shown to enhance functional and metabolic recovery of treated hearts compared with conventional preservation solutions.Go 5

The exact mechanism of the protective action of hyaluronidase is unknown. Although the enzymatic action of hyaluronidase on cardiac hyaluronidate may play a role, it is possible that hyaluronidase stimulates the release of the endogenous vascular protectant, nitric oxide. Nitric oxide prevents platelet adhesionGo 6 and aggregation,Go 7 promotes platelet disaggregation in the vasculature,Go 7 and inhibits leukocyte adhesionGo 8 and aggregationGo 9 and neutrophil superoxide anion production.Go Go 10,11 If hyaluronidase stimulates the endogenous release of nitric oxide, this could be a mechanism of the cardioprotective action of the compound. The purpose of these experiments was to determine whether hyaluronidase can induce the release of nitric oxide from the coronary endothelium.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Animal preparation
Heartworm-free mongrel dogs (25-30 kg) of either sex were anesthetized with pentobarbital sodium (30 mg/kg intravenous injection; Fort Dodge Laboratories, Inc, Fort Dodge, Iowa) and exsanguinated. Each dog's chest was opened quickly, and the heart was harvested and immersed in cool, oxygenated physiologic salt solution of the following composition (in millimoles per liter): NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.22; CaCl2, 2.5; NaHCO3, 25.0; calcium-ethylenediaminetetraacetic acid, 0.016; and glucose, 11.1 (control solution). The procedures and handling of the animals were reviewed and approved by the Institutional Animal Care and Use Committee of the Mayo Foundation.

In vitro experiments
The left circumflex coronary artery was carefully dissected free of connective tissue and placed in the control solution. Segments (4-5 mm long) of blood vessel were prepared; great care was taken not to touch the intimal surface. In some segments, vascular smooth muscle function was tested without the influence of the endothelium; in these rings, the endothelium was removed by gently rubbing the intimal surface of the blood vessel with a pair of watchmaker's forceps. This procedure removes endothelium but does not affect the ability of vascular smooth muscle to contract or relax.Go Go 12,13

Coronary artery segments, with and without endothelium, were suspended in organ chambers (25 mL) filled with control solution maintained at 37°C and aerated with 95% oxygen and 5% carbon dioxide (pH = 7.4). Each ring was suspended by 2 stainless steel clips passed through the lumen. One clip was anchored to the bottom of the organ chamber, and the other was connected to a strain gauge to measure isometric force (Grass FTO3; Grass Instrument Company, Quincy, Mass). The rings were placed at the optimal point of their length-tension relationship by progressively stretching them until contraction to potassium ions (20 mmol/L), at each level of distention, was maximal. In all experiments, the presence or absence of endothelium was confirmed by finding the response to acetylcholine (10-6 mol/L) in rings contracted with potassium ions (20 mmol/L).Go Go 12,13 After optimal tension was achieved, the arterial segments were allowed to equilibrate for 30 to 45 minutes before administration of drugs.

Drugs
The following drugs were used: bovine testicular hyaluronidase (150 USP units) (Wyeth Laboratories Inc, Philadelphia, Pa); indomethacin (INN: indometacin), prostaglandin F2{alpha}, methylene blue (Sigma Chemical Company, St Louis, Mo); and L-arginine, D-arginine, NG-monomethyl-L-arginine (L-NMMA), and NG-nitro-L-arginine (L-NOARG) (Calbiochem, San Diego, Calif). All powdered drugs were prepared with distilled water except for indomethacin, which was dissolved in Na2CO3 (10–5 mol/L). Oxyhemoglobin was prepared by the method of Gillespie and Sheng.Go 14

In some experiments, the enzymatic function of hyaluronidase was inactivated by heat by boiling the compound for 30 minutes. The sodium bicarbonate in the control solution, in addition to aeration with 5% carbon dioxide, has a buffering action in the organ bath that keeps the pH at 7.4. Thus, the addition of hyaluronidase, L-NMMA, L-arginine, or D-arginine did not alter organ bath pH. Unless otherwise stated, all drug concentrations are expressed as final molar concentration in the organ chambers. The effect of hyaluronidase on vascular reactivity of arterial segments with and without endothelium was studied on untreated arteries (control) or on arterial segments that were treated with L-NMMA (10–5 mol/L), L-NMMA plus L-arginine (10–4 mol/L), L-NMMA plus D-arginine (10–4 mol/L), L-NOARG (10–5 mol/L), hemoglobin (2 x 10–6 mol/L), and methylene blue (10–5 mol/L). All blockers were added to the organ bath at least 15 minutes before the coronary artery contractions with prostaglandin F2{alpha} (2 x 10–6 mol/L). All experiments were performed in the presence of indomethacin (10–6 mol/L) to prevent the synthesis of endogenous prostanoids.

Data analysis
Results are expressed as mean ± standard error of the mean. In all experiments, "n" refers to the number of animals from which blood vessels were taken. In segments contracted with prostaglandin F2{alpha}, responses are expressed as percent change from the contracted levels. Statistical evaluation of data was performed by the Student t test for either paired or unpaired observations and by analysis of variance.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Hyaluronidase (3.0 to 15 USP units) produced concentration-dependent relaxation in canine coronary artery segments with endothelium, but no change occurred in tension of arterial segments without endothelium(Figs 1,2, and3). Endothelium-dependent relaxation to hyaluronidase was blocked by pretreatment of arterial segments with L-NMMA (10–5 mol/L) or L-NOARG (10–5 mol/L), 2 competitive inhibitors of nitric oxide synthesis from L-arginineGo Go 15,16 (n = 6, each group; P < .05)(Figs 1Go,2Go, and3Go). The inhibitory effect of L-NMMA could be reversed by the addition of L-arginine (10–4 mol/L) but not D-arginine (10–4 mol/L) (n = 6, each group; P < .05)(Figs 1Go,2Go, and3Go). Endothelium-dependent relaxation to hyaluronidase also was blocked by pretreating vascular segments with methylene blue (10–6 mol/L), an inactivator of soluble guanylate cyclaseGo Go 17,18 (n = 6, P < .05)(Fig 3Go). The addition of hemoglobin (10–6 mol/L), a scavenger of the nitric oxide radical,Go 18 also abolished endothelium-dependent relaxation to hyaluronidase (n = 6, P < .05)(Fig 3Go). Inactivation of the enzymatic activity of hyaluronidase by heat did not alter endothelium-dependent relaxation to the compound (data not shown).



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  		Fig. 1. Endothelium-dependent relaxation to hyaluronidase in canine coronary arteries (original traces). Blood vessel segments with endothelium were contracted with prostaglandin F2{alpha} (PGF2 alpha; 2 x 10–6 mol/L) and exposed to increasing amounts of hyaluronidase. Top trace, Control vasodilation; second trace, response to hyaluronidase in the presence of NG-monomethyl-L-arginine(L-NMMA); third trace, vasodilation to hyaluronidase in the presence of L-NMMA and L-arginine(L-Arg); bottom trace, response to hyaluronidase in the presence of L-NMMA and D-arginine(D-Arg).

 


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  		Fig. 2. Concentration-response curves to hyaluronidase in canine coronary arteries. Segments with and without endothelium were contracted with prostaglandin F2{alpha} (PGF2 alpha; 2 x 10–6 mol/L) and exposed to increasing concentrations of hyaluronidase. Values are expressed as mean ± standard error of mean; n = 6, each group. D-Arg, D-Arginine; L-Arg, L-arginine; L-NMMA, NG-monomethyl-L-arginine.

 


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  		Fig. 3. Maximal vasodilation to hyaluronidase in canine coronary artery segments with or without endothelium. Segments with and without endothelium were contracted with prostaglandin F2{alpha} (PGF2 alpha; 2 x 10–6 mol/L) and exposed to hyaluronidase (15 units) only (control) or in the presence of NG-monomethyl-L-arginine(L-NMMA), L-arginine(L-Arg), D-arginine(D-Arg), NG-nitro-L-arginine (NO-Arg), oxyhemoglobin (Hb), or methylene blue (n = 6, each group). Asterisk denotes significance from arterial segments with endothelium (P < .05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study demonstrates that hyaluronidase induces the release of the endogenous vasodilator nitric oxide from the arterial endothelium. In the endothelial cell, nitric oxide is synthesized from the basic amino acid L-arginine,Go 19 and nitric oxide synthesis can be blocked by modified forms of the amino acid.Go Go 15,16 Indeed, in this experiment, endothelium-dependent vasodilation to hyaluronidase is blocked by L-NMMA and L-NOARG. The specificity of the competitive inhibition of L-NMMA for L-arginine metabolism was highlighted by the fact that the action of L-NMMA is reversed by adding exogenous L-arginine but not D-arginine. In addition, endothelium-dependent vasodilation to hyaluronidase is blocked by hemoglobin, which is a scavenger of the nitric oxide radical.Go 18 Nitric oxide activates soluble guanylate cyclase in the vascular smooth muscle to effect vasodilation.Go Go 17,18 Methylene blue is an inhibitor of soluble guanylate cyclase in the vascular smooth muscle.Go Go 17,18 When methylene blue is added to the organ chamber, endothelium-dependent vasodilation to hyaluronidase is abolished. Endothelium-dependent vasodilation to hyaluronidase does not involve epoprostenol (Prostacyclin) or other prostanoids, because vasodilation occurs even after inhibition of cyclooxygenase by indomethacin.

Enzymatic activity of hyaluronidase is not required to produce vasodilation, because inactivation of the enzyme compound by heat has no effect on vasodilation. This finding raises the possibility of a hyaluronidase receptor on the endothelial cell surface that mediates nitric oxide release. Another potential mechanism is interference with degradation of nitric oxide by the protein.

In 1959, Martins de Oliveira, Carballo, and ZimmermanGo 2 reported that in patients with myocardial infarctions hyaluronidase administration reduces ST-segment elevation. Subsequently, Maroko and associatesGo 1 demonstrated that hyaluronidase infusion diminishes myocardial necrosis in a canine model of acute coronary artery occlusion. Recently, investigators examining myocardial preservation found that even small concentrations of hyaluronidase added to preservation solutions improve functional and metabolic recovery in donor hearts treated with the compound.Go 5

Although some investigators attribute the protective action of hyaluronidase to its ability to decrease myocardial edema and augment cardiac lymphatic drainage,Go Go 3,4 the present study indicates that an additional mechanism could be hyaluronidase-stimulated release of nitric oxide.

Nitric oxide is an endogenous vascular protectant released by the intima. Nitric oxide prevents vasospasm and thrombosis in the coronary artery by inducing vasodilation, preventing platelet adhesionGo 6 and aggregation,Go 7 and promoting platelet disaggregationGo 20 in the vasculature. Nitric oxide also protects against cell-mediated reperfusion injury by preventing leukocyte adhesion to the intimaGo 8 and inhibiting leukocyte aggregation.Go 9 In addition, nitric oxide inactivates superoxide radicals produced by leukocytes.Go Go 10,11 Nitric oxide, having an unpaired electron, can accept electrons and thereby inactivate or scavenge O·2–. Thus, nitric oxide also acts as an endogenous defense against oxygen-derived free radicals.

By stimulating the endogenous release of nitric oxide, hyaluronidase activates a powerful system to protect the vasculature and the myocardium from the deleterious primary and secondary effects of ischemia and reperfusion. The present experiments lend support to the use of hyaluronidase as a vascular protectant during coronary reperfusion or for myocardial preservation.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Maroko PR, Libby P, Bloor CM, Sobel BE, Braunwald E. Reduction by hyaluronidase of myocardial necrosis following coronary artery occlusion. Circulation 1972;46:430-7.[Abstract/Free Full Text]
  2. Martins de Oliveira J, Carballo R, Zimmerman HA. Intravenous injection of hyaluronidase in acute myocardial infarction: preliminary report of clinical and experimental observations. Am Heart J 1959;57:712-22.[Medline]
  3. Taira A, Uehara K, Fukuda S, Takenada K, Koga M. Active drainage of cardiac lymph in relation to reduction in size of myocardial infarction: an experimental study. Angiology 1990;41:1029-36.
  4. Repa I, Garnic JD, Hollenberg NK. Myocardial infarction treated with two lymphagogues, calcium dobesilate (CLS 2210) and hyaluronidase: a coded, placebo-controlled animal study. J Cardiovasc Pharmacol 1990;16:286-91.[Medline]
  5. Fischer JH, Jeschkeit S. Minimal amounts of hyaluronidase in HTK or UW solution substantially improve the recovery of preserved hearts. Transplant Int 1996;9(Suppl 1):S442-6.
  6. Radomski MW, Palmer RM, Moncada S. The role of nitric oxide and cGMP in platelet adhesion to vascular endothelium. Biochem Biophys Res Commun 1987;148:1482-9.[Medline]
  7. Radomski MW, Palmer RM, Moncada S. Comparative pharmacology of endothelium-derived relaxing factor, nitric oxide and prostacyclin in platelets. Br J Pharmacol 1987;92:181-7.[Medline]
  8. Kubes P, Suzuki M, Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A 1991;88:4651-5.[Abstract/Free Full Text]
  9. McCall T, Whittle BJR, Boughton-Smith NK, Moncada S. Inhibition of FMLP-induced aggregation of rabbit neutrophils by nitric oxide (abstract). Br J Pharmacol 1988:95:517P.
  10. Clancy RM, Leszczynska-Piziak J, Abramson SB. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J Clin Invest 1992;90:1116-21.
  11. Rubanyi GM, Ho EH, Cantor EH, Lumma WC, Botelho LH. Cytoprotective function of nitric oxide: inactivation of superoxide radicals produced by human leukocytes. Biochem Biophys Res Commun 1991;181:1392-7.[Medline]
  12. Pearson PJ, Schaff HV, Vanhoutte PM. Acute impairment of endothelium-dependent relaxations to aggregating platelets following reperfusion injury in canine coronary arteries. Circ Res 1990;67:385-93.[Abstract/Free Full Text]
  13. Pearson PJ, Schaff HV, Vanhoutte PM. Long-term impairment of endothelium-dependent relaxations to aggregating platelets after reperfusion injury in canine coronary arteries. Circulation 1990;81:1921-7.[Abstract/Free Full Text]
  14. Gillespie JS, Sheng H. Influence of haemoglobin and erythrocytes on the effects of EDRF, a smooth muscle inhibitory factor, and nitric oxide on vascular and non-vascular smooth muscle. Br J Pharmacol 1988;95:1151-6.[Medline]
  15. Moore PK, al-Swayeh OA, Chong NW, Evans RA, Gibson A. L-NG-nitro arginine (L-NOARG), a novel, L-arginine–reversible inhibitor of endothelium-dependent vasodilation in vitro. Br J Pharmacol 1990;99:408-12.[Medline]
  16. Rees DD, Palmer RM, Hodson HF, Moncada S. A specific inhibitor of nitric oxide formation from L-arginine attenuates endothelium-dependent relaxation. Br J Pharmacol 1989;96:418-24.[Medline]
  17. Feelisch M, Noack EA. Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase. Eur J Pharmacol 1987;139:19-30.[Medline]
  18. Gruetter CA, Gruetter DY, Lyon JE, Kadowitz PJ, Ignarro LJ. Relationship between cyclic guanosine 3':5'-monophosphate formation and relaxation of coronary arterial smooth muscle by glyceryl trinitrate, nitroprusside, nitrite and nitric oxide: effects of methylene blue and methemoglobin. J Pharmacol Exp Ther 1981;219:181-6.[Abstract/Free Full Text]
  19. Palmer RM, Rees DD, Ashton DS, Moncada S. L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 1988;153:1251-6.[Medline]
  20. Furlong B, Henderson AH, Lewis MJ, Smith JA. Endothelium-derived relaxing factor inhibits in vitro platelet aggregation. Br J Pharmacol 1987;90:687-92.[Medline]
Received for publication Dec 17, 1999 Revisions requested March 16, 2000; revisions received May 7, 2000. Accepted for publication May 25, 2000.



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