Stimulation of rat uterine collagen synthesis by relaxin

Immature, ovariectomized, estrogen-primed rats respond to the administration of porcine relaxin by an increase in the incorporation of labeled amino acids ([14C]leucine, [14C]phenylalanine, [3H]proline) into uterine proteins in vitro. The maximum response occurs about 12 hr after a single injection of 0.1 mg relaxin in benzopurpurine 4B solution; subsequently, the relaxin effect declines but is still apparent after 24 hr. Smaller, but still significant increases in incorporation rates can be induced by relaxin in the absence of estrogen priming. Uterine collagen synthesis, as indicated by the incorporation of [3H]proline and its conversion to hydroxyproline, appears to be a primary target of the relaxin stimulus, since the effect of relaxin upon proline incorporation into uterine collagen is significantly greater than its effect upon labeling of noncollagen protein.     

Effects of porcine relaxins upon uterine hypertrophy and protein metabolism in mice

Two variant forms of porcine relaxin (B and C) are active in producing relaxation of the guinea pig pubic symphysis and in effecting uterine growth in rats. Only relaxin B, however, is active in the mouse pubic ligament assay. These two hormones were compared in mice for their effects upon uterine growth and incorporation of radioactively labeled proline into soluble protein and collagen in vitro and in vivo. Both relaxin B and relaxin C produced an early (3-hr) elevation in in vitro protein synthesis and a later (6-hr) increase in collagen incorporation of proline at the time when the uterotrophic effect was maximal. In vivo effects of relaxin C on the uterus were in some cases greater than relaxin B in contrast to the complete inactivity of the former upon the pubic ligament of the mouse. These findings suggest a high degree of tissue specificity for relaxin stimulation, a variability in responsiveness among tissues in the same animal, and perhaps a primary role of relaxin in uterine function with pelvic relaxation representing a secondary function which has developed in certain species.     

Physiologic role of relaxin on mammary gland growth in rats

The role of relaxin in stimulating growth of the mammary gland was assessed in ovariectomized and intact male rats for a period of 20 days. In addition to relaxin alone, the ovarian mammogenic hormones estradiol and progesterone were used in combination with relaxin and with each other to evaluate responses of mammae. Indices for mammary growth included wet weight, dry fat-free tissue, DNA, RNA, total protein, and collagen. Quantitative estimates of DNA and collagen represented the best indicators of parenchymal and stromal growth, respectively. Because changes in body weights were significantly different among hormonally administered groups, these were included as well. In Ovariectomized young rats, relaxin alone and in combination with estradiol and progesterone increased all indices significantly (P less than 0.01). The collagenous portion of total protein was high for the group receiving relaxin alone (62%) compared with the control group (46%). Relaxin administered along with estradiol and progesterone increased collagen accumulation to 73%, compared with 54% in the estradiol + progesterone group. Relaxin did not significantly increase growth indices when administered to male rats at 10 and 20 micrograms/day, while 30 micrograms stimulated a significant increase in total protein (P less than 0.05), suggesting that 30 micrograms of relaxin/day may be considered the basal concentration needed to induce a physiologic response in males. Relaxin induced a growth effect on mammae by synergizing with progesterone and estradiol in order to stimulate parenchymal proliferation, as noted by a DNA increase, and to increase stromal distensibility of the mammary pad by invoking accumulation of collagen and total protein in substituting for mammary adipose tissue.     

Extracellular matrix metabolism by chondrocytes. VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[3H]proline into hydroxyproline and [3H]acetate into glycosaminoglycans, was shown to be depressed by 58% and 39%, respectively, by the addition of exogenous proteoglycan at a concentration of 10 mg/ml growth media. The incorporation of L-[3H]proline into acid-insoluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity on the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-translational modification of collagen and proteoglycan.     

Relaxin and beta-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by beta-estradiol. We postulated that relaxin and beta-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, beta-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered beta-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 microg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. Beta-estradiol, relaxin, or beta-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or beta-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by beta-estradiol, relaxin, or beta-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

The in vivo inhibition of collagen synthesis and the reduction of prolyl hydroxylase activity by 3,4-dehydroproline.

Various proline analogs and iron chelators were tested for their effect on collagen formation which occurs in the uterus of the immature rat following the administration of estradiol-17β. dl-3,4-Dehydroproline, l-α-azetidine-2-carboxylic acid and l-pyroglutamic acid reduced the estradiol-17β stimulated formation of hydroxyproline which occurs in the uterus following administration of the hormone while l-thiazolidine-4-carboxylic acid was without effect on this response. The activity of the d- and l-isomers of 3,4-dehydroproline was compared with the racemic mixture; the l-isomer was twice as active as the latter, while the d-isomer was only half as active. l-3,4-Dehydroproline was approximately four times as potent as l-α-azetidine-2-carboxylic acid, the second most active analog of those tested. dl-3,4-Dehydroproline inhibited the incorporation of l-[¹⁴C]proline into the proline and hydroxyproline of uterine collagen; it also inhibited the incorporation of [¹⁴C]glycine into collagen while having less effect on the incorporation of these amino acids into noncollagen protein. These results indicate dl-3,4-dehydroproline is a fairly specific and potent inhibitor of collagen formation in vivo.These observations indicate that dl-3,4-dehydroproline reduces the hydroxylation of prolyl residues in collagen. Presumably, this occurs in part due to the incorporation of the analog into the collagen molecule in place of proline. It is probably also related to a reduction of prolyl hydroxylase activity which can be demonstrated in the tissues of animals treated with 3,4-dehydroproline. A significant reduction of prolyl hydroxylase activity was shown to persist in the uterus, lung, and heart for approximately 24 h following a single intraperitoneal dose of dl-3,4-dehydroproline (200 mg/kg).     

Relaxin and β-estradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss

Relaxin, a 6-kDa polypeptide hormone, is a potent mediator of matrix turnover and contributes to the loss of collagen and glycosaminoglycans (GAGs) from reproductive tissues, including the fibrocartilaginous pubic symphysis of several species. This effect is often potentiated by β-estradiol. We postulated that relaxin and β-estradiol might similarly contribute to the enhanced degradation of matrices in fibrocartilaginous tissues from synovial joints, which may help explain the preponderance of diseases of specific fibrocartilaginous joints in women of reproductive age. The objective of this study was to compare the in vivo effects of relaxin, β-estradiol, and progesterone alone or in various combinations on GAG and collagen content of the rabbit temporomandibular joint (TMJ) disc fibrocartilage, knee meniscus fibrocartilage, knee articular cartilage, and the pubic symphysis. Sham-operated or ovariectomized female rabbits were administered β-estradiol (20 ng/kg body weight), progesterone (5 mg/kg), or saline intramuscularly. This was repeated 2 days later and followed by subcutaneous implantation of osmotic pumps containing relaxin (23.3 μg/kg) or saline. Tissues were retrieved 4 days later and analyzed for GAG and collagen. Serum relaxin levels were assayed using enzyme-linked immunosorbent assay. Relaxin administration resulted in a 30-fold significant (p < 0.0001) increase in median levels (range, approximately 38 to 58 pg/ml) of systemic relaxin. β-estradiol, relaxin, or β-estradiol + relaxin caused a significant loss of GAGs and collagen from the pubic symphysis and TMJ disc and of collagen from articular cartilage but not from the knee meniscus. Progesterone prevented relaxin- or β-estradiol-mediated loss of these molecules. The loss of GAGs and collagen caused by β-estradiol, relaxin, or β-estradiol + relaxin varied between tissues and was most prominent in pubic symphysis and TMJ disc fibrocartilages. The findings suggest that this targeted modulation of matrix loss by hormones may contribute selectively to degeneration of specific synovial joints.     

Extracellular matrix metabolism by chondrocytes: VI. Concomitant depression by exogenous levels of proteoglycan of collagen and proteoglycan synthesis by chondrocytes

The synthesis of collagen and proteoglycans by cultured chondrocytes, as measured by the incorporation of L-[³H]proline into hydroxyproline and [³H]acetate into glycosaminoglycans, was shown to be depressed by 59% and 39%, respectively, by the addition of exogenous proteoglycan at a concetration of 10 mg/ml growth media. The incorporation of L-[³H]proline into acid-in-soluble protein remained unaltered in the presence of the proteoglycan. It was concluded that the effect was depressing the activity of the enzymatic steps, associated with the endoplasmic reticulum, which are responsible for the post-traslational modification of collagen and proteoglycan.     

Glucocorticoids stimulate collagen and noncollagen protein synthesis in cultured vascular smooth muscle cells

The effect of glucocorticoids on collagen synthesis was examined in cultured bovine aortic smooth muscle (BASM) cells. BASM cells treated with 0.1 microM dexamethasone during their proliferative phase (11 d) were labeled with [3H]proline for 24 h, and the acid-precipitable material was incubated with bacterial collagenase. Dexamethasone produced an approximate twofold increase in the incorporation of proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) in the cell layer and medium. The stimulation was present in both primary mass cultures and cloned BASM. An increase in CDP and NCP was detected at 0.1 nM, while maximal stimulation occurred at 0.1 microM. Only cells exposed to dexamethasone during their log phase of growth (1-6 d after plating) showed the increase in CDP and NCP when labeled 11 d after plating. The stimulatory effect was observed in BASM cells treated with the natural bovine glucocorticoid, cortisol, dexamethasone, and testosterone, but was absent in cells treated with aldosterone, corticosterone, cholesterol, 17 beta-estradiol, and progesterone. The increase in CDP and NCP was absent in cells treated with the inactive glucocorticoid, epicortisol, and totally abolished by the antagonist, 17 alpha-hydroxyprogesterone, suggesting that the response was mediated by specific cytoplasmic glucocorticoid receptors. Dexamethasone-treated BASM cells showed a 4.5-fold increase in the specific activity of intracellular proline, which was the result of a twofold increase in the uptake of proline and depletion of the total proline pool. After normalizing for specific activity, dexamethasone produced a 2.4- and 2.8-fold increase in the rate of collagen and NCP synthesis, respectively. Cells treated with dexamethasone secreted 1.7- fold more collagen protein in 24 h compared to control cultures. The BASM cells secreted 70% Type I and 30% Type III collagen into the media as assessed by two-dimensional gel electrophoresis. The ratio of these two types was not altered by dexamethasone. The results of the present study demonstrate that glucocorticoids can act directly on vascular smooth muscle cells to increase the synthesis and secretion of collagen and NCP.     

16,16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16,16 dimethyl prostaglandin E2 (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more 14C-proline and produced more 14C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10(-10) M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG: while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.     

Studies in vivo on the biosynthesis of collagen and elastin in ascorbic acid-deficient guinea pigs. Evidence for the formation and degradation of a partially hydroxylated collagen

1. After the administration of l-[G-(3)H]proline to guinea pigs deprived of ascorbic acid for increasing periods of time, the specific radioactivities of proline and hydroxyproline in skin collagen and aortic elastin were determined at various time-intervals after administration of the labelled compound with a view to studying the formation and degradation of collagen and elastin both deficient in hydroxyproline. 2. As judged from the incorporation of radioactivity into elastin proline, elastin synthesis was not decreased in the ascorbic acid-deficient animals. There was however, a rapid decline in the specific radioactivity of elastin hydroxyproline. The proline/hydroxyproline specific-radioactivity ratio was approx. 1.5:1 after 6 days and 20:1 after 12 days of ascorbic acid deprivation, in contrast with the ratio of 1:1 in controls. The results suggested that the effect of ascorbic acid deficiency on elastin biosynthesis could be regarded as simply an elimination of hydroxylation of elastin proline with the formation and retention of a polymer increasingly deficient in hydroxyproline. 3. Collagen proline and hydroxyproline specific radioactivities were derived from material that was soluble in hot trichloroacetic acid, non-diffusible and collagenase-degradable. In contrast with elastin, there was a rapid decline in the specific radioactivity of proline as well as hydroxyproline in collagen from the ascorbic acid-deficient animals. However, the proline/hydroxyproline specific-radioactivity ratio in all samples from scorbutic animals was consistently slightly above 1:1. The results suggest the appearance in place of collagen, but in rapidly diminishing amounts, of a partially hydroxylated collagen in which the degree of hydroxylation may be decreased only by approx. 10%. 4. Incorporation of radioactivity into the diffusible hydroxyproline in skin remained relatively high despite the rapid decline in the incorporation of radioactivity into collagen. This observation is interpreted as indicative of an increasing degree of degradation of partially hydroxylated collagen to diffusible peptides. An alternative explanation might be that partially hydroxylated peptides are released to an increasing extent from ribosomes before they attain a length at least sufficient to render them non-diffusible. In either case it implies the accumulation in scurvy of low-molecular-weight peptides enriched in proline and deficient in hydroxyproline and could explain the failure to accumulate a high-molecular-weight collagen deficient in hydroxyproline. 5. It is thought, however, that, in addition, an inhibition of ribosomal amino acid incorporation leading to decreased synthesis of partially hydroxylated collagen may also occur, perhaps secondarily to impaired hydroxylation.     

Rapid intracellular degradation of newly synthesized collagen by bone cells. Effect of dichloromethylenebisphosphonate

Experiments were carried out to determine whether bone cells isolated from rat calvaria degrade newly synthesized collagen intracellularly prior to secretion and to assess the effect of dichloromethylenebisphosphonate, a compound shown to stimulate collagen synthesis during this event. The findings indicate that isolated bone cells grown in culture degraded a proportion (average 16%) of newly synthesized collagen prior to secretion. This process was markedly reduced by exposure to dichloromethylenebisphosphonate in a dose-related manner. Concomitantly with the observed decrease of degradation, an increase of collagen synthesis was detected as determined by the incorporation of [3H]proline into collagenase-digestible proteins or by the conversion of [3H]proline into [3H]hydroxyproline. No similar enhancement on total non-collagenous protein synthesis was evident. Dichloromethylenebisphosphonate did not influence the extracellular degradation of collagen. Although the reduction in intracellular degradation accounted only for part of the bisphosphonate mediated increase in net collagen synthesis, it is conceivable that the rate of collagen synthesis is regulated, at least in part, by mechanisms that modulate the level of intracellular degradation.     

Effect of oestradiol-17 beta on ovarian and serum concentrations of relaxin during the second half of pregnancy in the rat

Immunoactivity concentrations of ovarian relaxin, serum relaxin and serum progesterone were determined from Day 12 through Day 18 of pregnancy in rats treated with oil or oestradiol-17 beta after hysterectomy or hypophysectomy and hysterectomy on Day 12. Relaxin and progesterone concentrations increased between Days 12 and 18 in sham-operated rats but failed to increase or declined in oil-treated hysterectomized or hypophysectomized-hysterectomized animals. Oestradiol treatment increased serum concentrations of relaxin and progesterone in hypophysectomized-hysterectomized rats on Day 15 and increased the concentrations of ovarian and serum relaxin and serum progesterone in hysterectomized rats on Day 18. These data are consistent with the concept that placental support for the promotion and maintenance of relaxin and progesterone concentrations from Day 12 through Day 18 may be mediated, at least in part, through a common mechanism(s) which involves oestradiol.     

Effect of estradiol and relaxin on collagen and non-collagen protein synthesis by mammary fibroblasts

In order to determine the effect of relaxin and estradiol on collagen and noncollagen synthesis by mammary gland fibroblasts, fibroblasts isolated from guinea pig mammary glands were grown on plastic or Cytodex-3 collagen coated microcarriers. On plastic, estradiol (600 pg/ml) increased the incorporation of tritiated glycine into collagen. Non-collagenous protein synthesis was increased at all concentrations of estradiol, but it was greatest with 200 pg/ml estradiol. On Cytodex-3, 400 pg/ml estradiol increased the synthesis of collagen and non-collagenous protein. Relaxin (1 microgram/ml) did not affect collagen synthesis but decreased the synthesis of non-collagenous protein.     

Effect of relaxin on the phenotype of collagens synthesized by cultured rabbit chondrocytes

The effect of porcine relaxin on rabbit articular and growth plate chondrocytes in primary culture was investigated by measurement of total collagen production and analysis of the phenotypes of newly synthesized collagen chains. A 24-h treatment of monolayer articular and multilayer growth plate chondrocytes with 2 micrograms per ml relaxin had no effect on total DNA and did not significantly modify the amount of [3H]proline-labelled collagen chains secreted by the cells. However, polyacrylamide gel electrophoresis demonstrated relevant modifications in relaxin treated chondrocytes. A significant increase was observed in the proportion of type III collagen and in the intensity of the band corresponding to alpha 2I chains. Two-dimensional peptide mapping of CNBr-cleaved molecules indicated that the band that was identified as alpha 1II on monodimensional gels contained a significant proportion of alpha 1I collagen chains, as demonstrated by the presence of alpha 1I cyanogen bromide-digested peptides. The intensity of this band was increased by relaxin treatment. Furthermore, total RNA analysis by slot blot and Northern blot techniques showed a dose-dependent stimulation of alpha 1I and alpha 1III mRNA levels after incubation with increased relaxin concentrations, but no change in the amount of alpha 1II mRNA. These results suggested that when added to cartilage cells in vitro, relaxin modulated the expression of type I, type II and type III collagen genes by amplifying the dedifferentiation process.     

Regional differences in water content,collagen content,and collagen degradation in the cervix of nonpregnant cows

The cow could be a suitable model for studies concerning functional changes of the cervix. However, as in many species, the bovine cervix becomes softer in texture during the follicular phase of the estrous cycle compared to the luteal phase. In the present study, we explored if changes in the collagen network take place that could be responsible for this phenomenon and if regional differences in water content, collagen content, and collagen degradation along the cross-sectional and longitudinal axes of the cervix were present. Two groups of nonpregnant animals with different progesterone status were studied. One group (n = 11) was under high progesterone influence, and the other group (n = 12) was under low progesterone influence. The water content was derived from the weight of the samples before and after lyophilization. The collagen content and the ratio of collagenous to noncollagenous proteins (hydroxyproline:proline ratio) were determined by performing amino acid analysis on hydrolyzed samples using high-performance liquid chromatography. Collagen denaturation was quantified with a colorimetric assay by determining the amount of hydroxyproline released from samples treated with alpha-chymotrypsine. The water content of the superficial layer of the submucosa was always significantly (P < 0.01) higher than the water content of the deep layer in the vaginal, mid, and uterine segments, but this was unrelated to the progesterone status of the animals. No effect of the tissue layers or of the progesterone status of the animals on the collagen content was observed, but an effect of segment was noted. The collagen content (mug/mg dry wt) in the vaginal segment of the cervix was significantly higher than in the mid (P < 0.05) and the uterine (P < 0.01) segments. The hydroxyproline:proline ratio showed the same pattern as the collagen content. The percentage of collagen denaturation in the superficial layer was always significantly (P < 0.01) higher than that in the deep layer, but no effect of the progesterone status or of the segment along the longitudinal axis was seen. It is concluded that regional differences in collagen biochemistry are present in the cervix of nonpregnant cows, which may account for the difference in firmness of different parts along the circular or the longitudinal axis of the cervix. However, differences in texture of the cervix between the two groups of cows could not be explained by differences in the collagen content, percentage of collagen denaturation, or water content.     

Metabolism of glomerular basement membrane in short- and long-term streptozotocin diabetic rats

The synthesis of glomerular basement membrane (GBM) total protein and collagen was assessed by two methods in vivo in normal and streptozotocin diabetic rats 4-6 weeks and 42-44 weeks after onset of hyperglycaemia, using L-[2, 3, 3H] proline as a radioactive precursor. The incorporation of tritiated proline into GBM hydroxyproline was used as a measure of collagen synthesis and that into proline as total protein synthesis. The basement membrane fractions from both short- and long-term diabetic rats attained much higher proline and hydroxyproline specific activities compared to normal GBM proline and hydroxyproline specific activities. Early insulin therapy with normalization of blood sugar levels in short-term (4-6 weeks) diabetic rats returned the abnormal increases in GBM total protein and collagen synthesis to normal. By contrast, poor glycaemic control with insulin did not prevent the increases in GBM protein synthesis. The results of the present study suggest that overall enhancement of GBM protein synthesis occurs in both short- and long-term streptozotocin diabetes. Early insulin therapy with normalization of blood sugar levels prevents this increase in GBM protein synthesis. Poor glycaemic control had no effect on abnormal GBM protein synthesis. This may be of potential significance in view of preventing chronic diabetic microvascular complications such as nephropathy.     

16, 16 Dimethyl prostaglandin E2 decreases the formation of collagen in fibrotic rat liver slices

The effect of 16, 16 dimethyl prostaglandin E₂ (DMPG) on fibrogenesis was studied in slices from normal and fibrotic rat liver. Rats received a cirrhogenic diet for seven months; supplemented controls received a diet with the deficient nutrients restored. Slices from fibrotic livers incorporated more ¹⁴C-proline and produced more ¹⁴C-hydroxyproline in TCA precipitable proteins than slices from control livers. DMPG (10^−10M) decreased the incorporation of labeled proline and the synthesis of labeled hydroxyproline in slices from fibrotic livers to the same extent, suggesting that DMPG did not affect the hydroxylation of proline per se. The magnitude of the DMPG induced decrease in labeled proline incorporation correlated with the hydroxyproline content in the liver (i.e. with increasing fibrosis there was a greater effect of DMPG; while in control rat liver slices, DMPG had no effect). DMPG did not change the size of the proline pool, its specific activity, or the activity of proline oxidase. We conclude that under these conditions of enhanced fibrogenesis, DMPG decreases the formation of collagen in vitro, possibly by lowering the incorporation of proline into collagen precursors. This may explain, at least in part, the inhibition of fibrogenesis by DMPG in vivo.