Turnover of Hyaluronan in the Microcirculation 1 , 2 ROLF K. REED and ULLA B. G. LAURENT
Introduction Our understanding of the physiology of the interstitial connective tissues and their role in fluid exchange has changed considerably during recent years (1,2). Previously, connective tissues were considered static supportive structures, with a low turnover and catabolism taking place exclusively inside the tissues. The development of sensitive analytic techniques during recent years (3, 4) has led to a series of investigations that have questioned this concept. These studies suggest that at least the most abundant glycosaminoglycanin skin, hyaluronan, is drained by lymph (5, 6) to be catabolized in local lymph nodes (7) and the liver (8). The present report provides a short review of the turnover of hyaluronan in skin, lung, and intestine and its possible implication for microcirculatory exchange. The focus will be on these three tissues since they contain more than 500/0 of the hyaluronan in the body (9), and they are also the tissues where hyaluronan turnover has been studied most extensively. For more detailed reviews on hyaluronan and its biologic function, see references 1 and 10 to 14. \.
Physicochemical Properties of Hyaluronan The hyaluronan moleculeis a linear chain containing repeating disaccharide units of glucuronic acid and N-acetyl-glucosamine. At physiologic pH each disaccharide carries one negative charge. A single molecule contains in the order of 10,000disaccharide units, and it has a molecular weight of several million Daltons. The physicochemical and physiologic properties of hyaluronan can be derived from its macromolecular structure, which in solution is that of a random coil. This occupies a volume that is 1,000times larger than that of the organic material (15),and neighboring molecules will entangle at concentrations exceeding 1 mg/ml (16).
Volume Exclusion Volume exclusion arises because two molecules cannot occupy the same space at the same time. Because of the large volume occupied by hyaluronan, hyaluronan solutions of 5 and 15 mg/ml exclude albumin from 25 and 75% of the total fluid volume, respectively (17). The exclusion phenomenon will decrease available volume, thereby increasing the concentration and colloid osmotic pressure of other macromolecules in their available volume, thus affecting transcapillary fluid transport. However, the capillaryinterstitial-lymphatic system is an open system with fluid influx across the capillary wall and out flux via the lymphatics. The effect of
SUMMARY Hyaluronan In skin, lung, and Intestine turns over within a few days and catabolism takes place locally In the tissues, In local lymph nodes, and In the liver. Hyaluronan will affect microcirculatory exchange through its influence on Interstitial volume exeluslon, hydraulic conductivity, and dlffusivity of macromolecules. Prolonged increase In interstitial fluid flux In Intestine has been shown to reduce the hyaluronan content, which in turn Increases hydraUlic conductivity and diffuAM REV RESPIR DIS 1992; 146:S37-S39 slvity of macromolecules.
altered exclusion by changing the amount of excluding substance is therefore not easily deductible. No in vivo experiments have been carried out to clarify the effect of altered exclusion, and it is difficult to see how such experiments could be performed. The effect of altered exclusionhas been studied in computer simulations (18, 19).These investigations suggest that increasing exclusionwilldecrease the time to attain a new steady-state interstitial volume and protein mass after a perturbation in capillary balance, whereas the final steady state level is not influenced (18, 19). The explanation to this observation seems to be that the increased exclusion decreases the interstitial protein content, and thereby the amount of protein that needs to be accumulated or drained from the interstitium after a perturbation in transcapillary balance. The other structural components of the interstitium have excludingproperties like hyaluronan. On a weight basis, collagen will exclude albumin from 1 to 4 mllg collagen (17). Because collagen is far more abundant than hyaluronan in most connective tissues, the overall contribution from collagen to exclusion seemsto be larger than that from hyaluronan (17, 20).
Hydraulic Conductivity The hydraulic conductivity of loose connective tissues ranges between 3 and 200·IO-s cmvs-mm Hg in umbilical cord and subcutis, respectively, whereas that of a 1% hyaluronan solution is 3·IO-scm 2/s·mmHg(15). Further, when comparing different tissues there is an inverse relationship between hyaluronan concentration and hydraulic conductivity of the tissue (21). In a recent review, Levick (22) analyzed the importance of the different structural components for hydraulic conductivity of the tissue using Darcy's law and concluded that neither hyaluronan and other glycosaminoglycans nor collagen can account for the hydraulic conductivity alone, that their combined effect was required to explain the experimental observations. Macromolecular Diffusion and Convection Hyaluronan will severely restrict macromolecular movement, and the diffusivity for albumin in a 1% hyaluronan solution is 50%
of that in water (23). A similar diffusivity is seenin human umbilical cord, whereasin subcutis the diffusivity of albumin is 75% of that in water (15). Thus, the physiologic importance of hyaluronan in microcirculatory exchange is through its action on the interstitial connective tissue where increasing hyaluronan concentration will increase interstitial volume exclusion, but decrease the hydraulic conductivity and diffusivity of macromolecules. Synthesis, Turnover, and Catabolism Synthesis and Turnover Hyaluronan is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugar residues to the reducing end of the chain (24). SKIN
Skin contains about half of the hyaluronan in the body and is therefore a major determinant of the total turnover of hyaluronan in the body (9). One gram of skin contains about 0.5 mg hyaluronan (9, 20). Because the interstitial fluid volume in skin is about 0.4 mllg wet weight (20), the concentration of hyaluronan is about 1 mg/ml, i.e., sufficient to cause entanglement (16). The half-life of hyaluronan in skin as measured on endogenously i·e-labeled hyaluronan is 2 to 4 days (25, 26). More recently, hyaluronan with molecular weight of 0.2 and 3 x 106 injected subcutaneously or intradermally have shown a half-life of removal of 12 to 16 h for tracer amounts (27, 28), i.e., even shorter than reported by Schiller and coworkers (25, 26). The two sets of data could be reconciled if there were two pools of hyaluronan, one "fixed," most likely constituted by hyaluronan bound in the pericellular coats of fibroblasts (29), or one bound
1 From the Department of Physiology, University of Bergen, Norway, and the Departments of Ophthalmology and Medical and Physiological Chemistry, University of Uppsala, Sweden. 2 Correspondence and requests for reprints should be addressed to Dr. Rolf K. Reed, Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
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to structural components of the tissue. The hyaluronan is then released into a "free" pool, which in turn is drained into lymph. According to this model the "fixed" pool of hyaluronan would have to constitute 750/0 and the "free" pool 250/0 of the total amount of hyaluronan in skin (27). The hyaluronan concentration in prenodal lymph from skin is 5 to 10 ug/rnl (30), i.e., 10 to 100 times less than the concentration that can be calculated from the tissue content and interstitial fluid volume (12). In experiments where interstitial fluid flux was increased, the hyaluronan concentration initially was constant when venous pressure was raised to 20 mm Hg (30). At higher venous pressures the concentration fell by 30% when lymph flow had been increased by raising venous pressure to between 30 and 40 mm Hg, corresponding to a fivefold increase in lymph flow.The combined effect of increased lymph flow and loweredconcentration increased the hyaluronan flux fourfold above control. Under normal conditions it was estimated that the lymph drained 1to 5% of the hyaluronan in the tissue per day. Burn injury to the skin followed by infusion therapy increased plasma hyaluronan 10fold above control (31), whereas infusion therapy alone resulted in a smaller increase in plasma hyaluronan (31). Because the hyaluronan concentration in lymph fell by 30% while lymph flow increased nearly fivefold as a consequence of burn injury, the increased influx to plasma was attributed to increased lymph flow (32), supporting the conclusion that hyaluronan is loosely bound in the skin. Catabolism in the Tissues Previously, it seemsto havebeen accepted that the catabolism of the large hyaluronan molecules took place in the tissues despite little direct evidence. The hyaluronidase isolated from skin (33) has an optimal pH of 5, with little enzyme activity above this pH. The optimal pH speaks against any extracellular activity of the enzymeunder normal conditions, but rather suggests a lysosomal activity. The local catabolism in skin has recently been estimated to 12 to 25% of the total turnover of hyaluronan in experiments using hyaluronan labeled with 125I-tyramine-cellobiose (28). Catabolism in Lymph Nodes As reported by Fraser and coworkers (7), as much as 90% of the hyaluronan reaching the popliteal lymph node in sheep could be catabolized in the node. Although no other studies have investigatedcatabolism in lymph nodes, the similar concentrations of hyaluronan in prenodal and postnodal lymph from several organs suggest that there might be a great variation between lymph nodes from different organs with respect to their capacity to degrade hyaluronan. The observation that 90% of the hyaluronan could be degraded in the lymph node most likely should be taken as a high or maximal value rather than as the amount typically catabolized in lymph nodes.
REED AND LAURENT
Catabolism in Liver The metabolic degradation of hyaluronan in the liver takes place in the liver endothelial cells and has been characterized in a series of studies during the last decade (reviewed in reference 8). When hyaluronan reaches plasma it is cleared with a half-life of 2 to 5 min (34, 35), but still the concentration in plasma is 10 to 100ng/ml (36), i.e., about 10 to 100times less than in lymph. The first step in the catabolism is a receptor-mediated endocytosis (8). The receptor recognizes an oligosaccharide sequence and, since this sequence is repeated along the chain, the endocytosis is more efficient in clearing large than in clearing small hyaluronan molecules from the bloodstream. This is reflected in a Ks« of 6 x 105 and 9 x 106 I!M for hyaluronan, with molecular weights of 4 X 105 and 6.4 x 106 , respectively (8). The hyaluronan molecule is catabolized to low molecular weight products and to carbon dioxide and water, which start to appear in the blood after 20 min (8). LUNG
The hyaluronan content in lungs from different species ranges between 15 and 150 ug/g wet weight (12). The hyaluronan in the lung is mostly localized to perialveolar and peribronchial tissue, but is also found in intraalveolar septa (37). Lebel and coworkers (37, 38) were the first to study the effect of increased interstitial fluid flux on hyaluronan flux from the interstitium. They measured the lymph flow and hyaluronan concentration in a chronically cannulated postnodallymphatic from the caudal mediastinal lymph node in sheep. Hyaluronan concentration in control averaged about 10ug/ml and did not change when interstitial fluid flux was increased by raising left atrial pressure to 25 mm Hg. Lymph flux of hyaluronan therefore increased to the same extent as lymph flow, i.e., five to six times above control when left atrial pressure was raised to about 25 mm Hg. The hyaluronan content in the lungs was 167 ug/g wettissue, and the lymphatic hyaluronan flux corresponded to about 2% of the total hyaluronan content per 24 h in control, but as much as 18% per 24 h at left atrial pressures of 25 mm Hg. The concentration in prenodal lymph from dog lungs has been reported to be 5 to 10 ug/rnl in control (39), i.e., similar to those reported in postnodal lymph from sheep (37, 38). Two different protocols were used by Townsleyand coworkers (39) to increase venous pressure, a stepwise increase or a one-step increase, on both occasions maintained for at least 4 h. Both protocols resulted in a twofold to fourfold increase in the hyaluronan concentration in lymph compared with that in control, and the hyaluronan flux increased tenfold that of the control. Thus, prolonged elevations of interstitial fluid flux will influence the turnover of hyaluronan. INTESTINE
The intestine contains 10 to 80 ug hyaluro-
nan per gram weight wet weight (9), and with an interstitial fluid volume of 0.15 mllg wet weight, the concentration is 0.5 mg/ml interstitial fluid (9). Hyaluronan is absent from the muscular layers of the intestine and is located in the lamina propria and in the lacteals of individual villi (40). The hyaluronan concentration in postnodal intestinal lymph from sheep and cats is 5 to 50 ug/ml (6, 41), i.e., a larger range and higher average concentrations than for most other organs studied. The intestinal interstitium provides a hindrance to transport of macromolecules like any other connective tissue, but in addition it provides a special problem since chylomicrons with diameters of 5 to 10,000 A will have to traverse the interstitium (42). Loose connectivetissues have been estimated to have "pores" for macromolecular transport with a size of 150 to 250 A (15), which seems insufficient to allow the passage of the large chylomicrons, Although no data are available specifically for the size of such "pores" in the connective tissue in the intestine, it seems plausible to expect properties similar to those in other loose connective tissues. Despite the small sizeof the channels seemingly available for the transport of chylomicrons, these appear in lymph and in the thoracic duct within 30 min after intestinal fat administration (43, 44).During this time the fat has been absorbed by the intestinal epithelium, chylomicrons have been formed on the interstitial side and transported through the connective tissue to reach the lymphatics. A potential mechanism to increase the size of the "pores" could be by washing out the hyaluronan and thereby increasing the size of the transport channels. Twoseriesof experiments on isolated and perfused cat ileal loops seem to confirm this hypothesis. When interstitial fluid flux through the intestinal interstitium was increased by elevating local venous pressure, the hyaluronan concentration in lymph initially increased and subsequently fell to very low levels (less than 5 ug/rnl as compared with a mean control of 20 ug/ml). After 4 h of increased interstitial fluid flux, the hyaluronan content in the intestine had been reduced to 40% of the control value (41). In a second series of experiments,intestinal administration of oleic acid and taurocholate resulted in a nearly threefold increase in lymphatic flux of hyaluronan (45). Taken together with the previous study, it supports the conclusion that interstitial hyaluronan can be washed out to an extent that promotes transport of macromolecules through the intestinal interstitium. Interaction with Cells Hyaluronan receptors have been demonstrated on fibroblasts and 3T3 cells, and they have a role in cell locomotion and homing of lymphocytes (46). Recently, hyaluronan receptors have also been reported on vascular endothelial cells (47). The investigators suggested that the receptors have a role during angiogenesis, but no physiologic role for the receptors for microvascular function has
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presently been demonstrated. Finally, it should be noted that hyaluronan decreases the viscosity of erythrocytes (48).
References 1. Laurent TC. Structure, function and turnover of the extracellular matrix. Adv Microcirc 1987; 13:15-34. 2. Fraser JRE, Laurent TC. Turnover and metabolism of hyaluronan. In: Evered D, Whelan J, eds. The biology of hyaluronan. Ciba Foundation Symposi urn no. 143.Chichester: John Wiley and Sons, 1989; 41-59. 3. Laurent UBG, Tengb1adA. Determination of hyaluronate in biological samples by a specific radioassay technique. Anal Biochem 1980; 109: 386-94. 4. Brandt R, nearer E, Asman I, Bucht A, Tengblad A. A convenient radiometric assay for hyaluronan. Acta Otolaryngol Suppi (Stockh) 1987; 442:31-5. 5. Laurent UBG, Laurent TC. On the origin of hyaluronate in blood. Biochem Int 1981; 2:195-9. 6. Tengblad A, Laurent UBG, Lilja K, et al. Concentration and relative molecular mass of hyaluronate in lymph and blood. Biochem J 1986; 236: 521-5. 7. Fraser JRE, Kimpton WG, Laurent TC, Cahill RNP, Vakakis N. Uptake and degradation of hyaluronan in lymphatic tissue. Biochem J 1988; 256:153-8. 8. Smedsred B, Pertoft H, Gustafson S, Laurent TC. Scavenger functions of the liver endothelial cell. Biochem J 1990; 266;313-27. 9. Reed RK, Lilja K, Laurent TC. Hyaluronan in the rat with special reference to the skin. Acta Physiol Scand 1988; 134:405-11." 10. Comper WD, Laurent TC. Physiological function of connective tissue polysaccharides. Physiol . Rev 1978; 58:255-315. 11. Evered D, Whelan J, eds. The biology of hyaluronan. Ciba Foundation Symposium no. 143. Chichester: John Wiley and Sons, 1989. 12. Laurent UBG, Reed RK. Turnover of hyaluronan in the tissues. Adv Drug Delivery Rev 1991; 7:237-56. 13. Drobnik J, ed. Hyaluronic acid in controlled drug delivery. Adv Drug Delivery Rev 1991; 7:221-308. 14. Laurent TC, Fraser JRE. Hyaluronan. FASEB J 1992; 6:2397-404. 15. Granger HJ. Physicochemical properties of the extracellular matrix. In: Hargens AR, ed. Tissue fluid pressure and composition. Baltimore: Williams and Wilkins, 1981; 43-61. 16. Bothner H, Wik O. Rheology of hyaluronate. Acta Otolaryngol Suppl (Stockh) 1987;442:25-30.
17. Bert JL, Pearce RH. The interstitium and microvascular exchange. In: Handbook of physiology. Section 2: The cardiovascular system. Vol. IV; Pt. 1: Renkin EM, Michel CC, eds. Microcirculation. Bethesda: American Physiological Society, 1984; 521-47. 18. Granger HJ, Laine GA, Barnes GE, LewisRE. Dynamics and control of transmicrovascular fluid exchange. In: Staub NC, Taylor AE, eds. Edema. New York: Raven Press, 1984; 189-228. 19. Reed RK, Bowen BD, Bert JL. Microvascular exchange and interstitial volume regulation in the rat: implications of the model. Am J Physiol1989; 257:H2081-91. 20. Reed RK, Lepsee S, Wiig H. Interstitial exclusion of albumin in rat dermis and subcutis in over- and dehydration. Am J Physiol 1989; 257:HI819-27. 21. Aukland K, NicolaysenG. Interstitial fluid volume: local regulatory mechanisms. Physiol Rev 1981; 61:556-643. 22. LevickJR. Flow through interstitium and other fibrous matrices. Q J Exp Physiol1987; 72:409-38. 23. Laurent TC, Bjork I, Pietruszkiewicz A, Persson H. On the interaction between polysaccharides and other macromolecules. Biochim Biophys Acta 1963; 78:351-9. 24. Prehm P. Hyaluronate is synthesizedat plasma membranes. Biochem J 1984; 220:597-600. 25. Schiller S, Mathews MB, Goldfaber L, Ludowieg J, Dorfman A. The metabolism of rnucopolysaccharides in animals. II. Studies in skin utilizing labeled acetate. J Bioi Chern 1955; 212:531-5. 26. Schiller S, Mathews MB, Cifonelli JA, Dorfman A. The metabolism of mucopolysaccharides in animals. III. Further studies on skin using C':'glucose, C'
Introduction Our understanding of the physiology of the interstitial connective tissues and their role in fluid exchange has changed considerably during recent years (1,2). Previously, connective tissues were considered static supportive structures, with a low turnover and catabolism taking place exclusively inside the tissues. The development of sensitive analytic techniques during recent years (3, 4) has led to a series of investigations that have questioned this concept. These studies suggest that at least the most abundant glycosaminoglycanin skin, hyaluronan, is drained by lymph (5, 6) to be catabolized in local lymph nodes (7) and the liver (8). The present report provides a short review of the turnover of hyaluronan in skin, lung, and intestine and its possible implication for microcirculatory exchange. The focus will be on these three tissues since they contain more than 500/0 of the hyaluronan in the body (9), and they are also the tissues where hyaluronan turnover has been studied most extensively. For more detailed reviews on hyaluronan and its biologic function, see references 1 and 10 to 14. \.
Physicochemical Properties of Hyaluronan The hyaluronan moleculeis a linear chain containing repeating disaccharide units of glucuronic acid and N-acetyl-glucosamine. At physiologic pH each disaccharide carries one negative charge. A single molecule contains in the order of 10,000disaccharide units, and it has a molecular weight of several million Daltons. The physicochemical and physiologic properties of hyaluronan can be derived from its macromolecular structure, which in solution is that of a random coil. This occupies a volume that is 1,000times larger than that of the organic material (15),and neighboring molecules will entangle at concentrations exceeding 1 mg/ml (16).
Volume Exclusion Volume exclusion arises because two molecules cannot occupy the same space at the same time. Because of the large volume occupied by hyaluronan, hyaluronan solutions of 5 and 15 mg/ml exclude albumin from 25 and 75% of the total fluid volume, respectively (17). The exclusion phenomenon will decrease available volume, thereby increasing the concentration and colloid osmotic pressure of other macromolecules in their available volume, thus affecting transcapillary fluid transport. However, the capillaryinterstitial-lymphatic system is an open system with fluid influx across the capillary wall and out flux via the lymphatics. The effect of
SUMMARY Hyaluronan In skin, lung, and Intestine turns over within a few days and catabolism takes place locally In the tissues, In local lymph nodes, and In the liver. Hyaluronan will affect microcirculatory exchange through its influence on Interstitial volume exeluslon, hydraulic conductivity, and dlffusivity of macromolecules. Prolonged increase In interstitial fluid flux In Intestine has been shown to reduce the hyaluronan content, which in turn Increases hydraUlic conductivity and diffuAM REV RESPIR DIS 1992; 146:S37-S39 slvity of macromolecules.
altered exclusion by changing the amount of excluding substance is therefore not easily deductible. No in vivo experiments have been carried out to clarify the effect of altered exclusion, and it is difficult to see how such experiments could be performed. The effect of altered exclusionhas been studied in computer simulations (18, 19).These investigations suggest that increasing exclusionwilldecrease the time to attain a new steady-state interstitial volume and protein mass after a perturbation in capillary balance, whereas the final steady state level is not influenced (18, 19). The explanation to this observation seems to be that the increased exclusion decreases the interstitial protein content, and thereby the amount of protein that needs to be accumulated or drained from the interstitium after a perturbation in transcapillary balance. The other structural components of the interstitium have excludingproperties like hyaluronan. On a weight basis, collagen will exclude albumin from 1 to 4 mllg collagen (17). Because collagen is far more abundant than hyaluronan in most connective tissues, the overall contribution from collagen to exclusion seemsto be larger than that from hyaluronan (17, 20).
Hydraulic Conductivity The hydraulic conductivity of loose connective tissues ranges between 3 and 200·IO-s cmvs-mm Hg in umbilical cord and subcutis, respectively, whereas that of a 1% hyaluronan solution is 3·IO-scm 2/s·mmHg(15). Further, when comparing different tissues there is an inverse relationship between hyaluronan concentration and hydraulic conductivity of the tissue (21). In a recent review, Levick (22) analyzed the importance of the different structural components for hydraulic conductivity of the tissue using Darcy's law and concluded that neither hyaluronan and other glycosaminoglycans nor collagen can account for the hydraulic conductivity alone, that their combined effect was required to explain the experimental observations. Macromolecular Diffusion and Convection Hyaluronan will severely restrict macromolecular movement, and the diffusivity for albumin in a 1% hyaluronan solution is 50%
of that in water (23). A similar diffusivity is seenin human umbilical cord, whereasin subcutis the diffusivity of albumin is 75% of that in water (15). Thus, the physiologic importance of hyaluronan in microcirculatory exchange is through its action on the interstitial connective tissue where increasing hyaluronan concentration will increase interstitial volume exclusion, but decrease the hydraulic conductivity and diffusivity of macromolecules. Synthesis, Turnover, and Catabolism Synthesis and Turnover Hyaluronan is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugar residues to the reducing end of the chain (24). SKIN
Skin contains about half of the hyaluronan in the body and is therefore a major determinant of the total turnover of hyaluronan in the body (9). One gram of skin contains about 0.5 mg hyaluronan (9, 20). Because the interstitial fluid volume in skin is about 0.4 mllg wet weight (20), the concentration of hyaluronan is about 1 mg/ml, i.e., sufficient to cause entanglement (16). The half-life of hyaluronan in skin as measured on endogenously i·e-labeled hyaluronan is 2 to 4 days (25, 26). More recently, hyaluronan with molecular weight of 0.2 and 3 x 106 injected subcutaneously or intradermally have shown a half-life of removal of 12 to 16 h for tracer amounts (27, 28), i.e., even shorter than reported by Schiller and coworkers (25, 26). The two sets of data could be reconciled if there were two pools of hyaluronan, one "fixed," most likely constituted by hyaluronan bound in the pericellular coats of fibroblasts (29), or one bound
1 From the Department of Physiology, University of Bergen, Norway, and the Departments of Ophthalmology and Medical and Physiological Chemistry, University of Uppsala, Sweden. 2 Correspondence and requests for reprints should be addressed to Dr. Rolf K. Reed, Department of Physiology, University of Bergen, Arstadveien 19, N-5009 Bergen, Norway.
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to structural components of the tissue. The hyaluronan is then released into a "free" pool, which in turn is drained into lymph. According to this model the "fixed" pool of hyaluronan would have to constitute 750/0 and the "free" pool 250/0 of the total amount of hyaluronan in skin (27). The hyaluronan concentration in prenodal lymph from skin is 5 to 10 ug/rnl (30), i.e., 10 to 100 times less than the concentration that can be calculated from the tissue content and interstitial fluid volume (12). In experiments where interstitial fluid flux was increased, the hyaluronan concentration initially was constant when venous pressure was raised to 20 mm Hg (30). At higher venous pressures the concentration fell by 30% when lymph flow had been increased by raising venous pressure to between 30 and 40 mm Hg, corresponding to a fivefold increase in lymph flow.The combined effect of increased lymph flow and loweredconcentration increased the hyaluronan flux fourfold above control. Under normal conditions it was estimated that the lymph drained 1to 5% of the hyaluronan in the tissue per day. Burn injury to the skin followed by infusion therapy increased plasma hyaluronan 10fold above control (31), whereas infusion therapy alone resulted in a smaller increase in plasma hyaluronan (31). Because the hyaluronan concentration in lymph fell by 30% while lymph flow increased nearly fivefold as a consequence of burn injury, the increased influx to plasma was attributed to increased lymph flow (32), supporting the conclusion that hyaluronan is loosely bound in the skin. Catabolism in the Tissues Previously, it seemsto havebeen accepted that the catabolism of the large hyaluronan molecules took place in the tissues despite little direct evidence. The hyaluronidase isolated from skin (33) has an optimal pH of 5, with little enzyme activity above this pH. The optimal pH speaks against any extracellular activity of the enzymeunder normal conditions, but rather suggests a lysosomal activity. The local catabolism in skin has recently been estimated to 12 to 25% of the total turnover of hyaluronan in experiments using hyaluronan labeled with 125I-tyramine-cellobiose (28). Catabolism in Lymph Nodes As reported by Fraser and coworkers (7), as much as 90% of the hyaluronan reaching the popliteal lymph node in sheep could be catabolized in the node. Although no other studies have investigatedcatabolism in lymph nodes, the similar concentrations of hyaluronan in prenodal and postnodal lymph from several organs suggest that there might be a great variation between lymph nodes from different organs with respect to their capacity to degrade hyaluronan. The observation that 90% of the hyaluronan could be degraded in the lymph node most likely should be taken as a high or maximal value rather than as the amount typically catabolized in lymph nodes.
REED AND LAURENT
Catabolism in Liver The metabolic degradation of hyaluronan in the liver takes place in the liver endothelial cells and has been characterized in a series of studies during the last decade (reviewed in reference 8). When hyaluronan reaches plasma it is cleared with a half-life of 2 to 5 min (34, 35), but still the concentration in plasma is 10 to 100ng/ml (36), i.e., about 10 to 100times less than in lymph. The first step in the catabolism is a receptor-mediated endocytosis (8). The receptor recognizes an oligosaccharide sequence and, since this sequence is repeated along the chain, the endocytosis is more efficient in clearing large than in clearing small hyaluronan molecules from the bloodstream. This is reflected in a Ks« of 6 x 105 and 9 x 106 I!M for hyaluronan, with molecular weights of 4 X 105 and 6.4 x 106 , respectively (8). The hyaluronan molecule is catabolized to low molecular weight products and to carbon dioxide and water, which start to appear in the blood after 20 min (8). LUNG
The hyaluronan content in lungs from different species ranges between 15 and 150 ug/g wet weight (12). The hyaluronan in the lung is mostly localized to perialveolar and peribronchial tissue, but is also found in intraalveolar septa (37). Lebel and coworkers (37, 38) were the first to study the effect of increased interstitial fluid flux on hyaluronan flux from the interstitium. They measured the lymph flow and hyaluronan concentration in a chronically cannulated postnodallymphatic from the caudal mediastinal lymph node in sheep. Hyaluronan concentration in control averaged about 10ug/ml and did not change when interstitial fluid flux was increased by raising left atrial pressure to 25 mm Hg. Lymph flux of hyaluronan therefore increased to the same extent as lymph flow, i.e., five to six times above control when left atrial pressure was raised to about 25 mm Hg. The hyaluronan content in the lungs was 167 ug/g wettissue, and the lymphatic hyaluronan flux corresponded to about 2% of the total hyaluronan content per 24 h in control, but as much as 18% per 24 h at left atrial pressures of 25 mm Hg. The concentration in prenodal lymph from dog lungs has been reported to be 5 to 10 ug/rnl in control (39), i.e., similar to those reported in postnodal lymph from sheep (37, 38). Two different protocols were used by Townsleyand coworkers (39) to increase venous pressure, a stepwise increase or a one-step increase, on both occasions maintained for at least 4 h. Both protocols resulted in a twofold to fourfold increase in the hyaluronan concentration in lymph compared with that in control, and the hyaluronan flux increased tenfold that of the control. Thus, prolonged elevations of interstitial fluid flux will influence the turnover of hyaluronan. INTESTINE
The intestine contains 10 to 80 ug hyaluro-
nan per gram weight wet weight (9), and with an interstitial fluid volume of 0.15 mllg wet weight, the concentration is 0.5 mg/ml interstitial fluid (9). Hyaluronan is absent from the muscular layers of the intestine and is located in the lamina propria and in the lacteals of individual villi (40). The hyaluronan concentration in postnodal intestinal lymph from sheep and cats is 5 to 50 ug/ml (6, 41), i.e., a larger range and higher average concentrations than for most other organs studied. The intestinal interstitium provides a hindrance to transport of macromolecules like any other connective tissue, but in addition it provides a special problem since chylomicrons with diameters of 5 to 10,000 A will have to traverse the interstitium (42). Loose connectivetissues have been estimated to have "pores" for macromolecular transport with a size of 150 to 250 A (15), which seems insufficient to allow the passage of the large chylomicrons, Although no data are available specifically for the size of such "pores" in the connective tissue in the intestine, it seems plausible to expect properties similar to those in other loose connective tissues. Despite the small sizeof the channels seemingly available for the transport of chylomicrons, these appear in lymph and in the thoracic duct within 30 min after intestinal fat administration (43, 44).During this time the fat has been absorbed by the intestinal epithelium, chylomicrons have been formed on the interstitial side and transported through the connective tissue to reach the lymphatics. A potential mechanism to increase the size of the "pores" could be by washing out the hyaluronan and thereby increasing the size of the transport channels. Twoseriesof experiments on isolated and perfused cat ileal loops seem to confirm this hypothesis. When interstitial fluid flux through the intestinal interstitium was increased by elevating local venous pressure, the hyaluronan concentration in lymph initially increased and subsequently fell to very low levels (less than 5 ug/rnl as compared with a mean control of 20 ug/ml). After 4 h of increased interstitial fluid flux, the hyaluronan content in the intestine had been reduced to 40% of the control value (41). In a second series of experiments,intestinal administration of oleic acid and taurocholate resulted in a nearly threefold increase in lymphatic flux of hyaluronan (45). Taken together with the previous study, it supports the conclusion that interstitial hyaluronan can be washed out to an extent that promotes transport of macromolecules through the intestinal interstitium. Interaction with Cells Hyaluronan receptors have been demonstrated on fibroblasts and 3T3 cells, and they have a role in cell locomotion and homing of lymphocytes (46). Recently, hyaluronan receptors have also been reported on vascular endothelial cells (47). The investigators suggested that the receptors have a role during angiogenesis, but no physiologic role for the receptors for microvascular function has
839
TURNOVER OF HYALURONAN IN THE MICROCIRCULATION
presently been demonstrated. Finally, it should be noted that hyaluronan decreases the viscosity of erythrocytes (48).
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