3H-proline Incorporation Research Articles

Aldosterone and myocardial fibrosis in heart failure.

Cardiac fibroblasts are known to have high affinity corticoid receptors for aldosterone and account for the accumulation of collagen within the interstitium of the rat myocardium in acquired and genetic hypertension. This interstitial fibrosis is an important determinant of pathologic hypertrophy in chronic heart failure. To examine the relationship between aldosterone and myocardial fibrosis, collagen volume fraction of the left and right ventricles were analyzed by videodensitometry of sirius red stained tissue in the following rat models: 2 kidney/1 clip model of renovascular hypertension; continuous aldosterone administration via osmotic minipumps (0.75 microgram/hour s.c.), or in each model of primary and secondary hyperaldosteronism with concomitant treatment with either low (20 mg/kg/day) or high doses (200 mg/kg/day) of s.c. spironolactone for 8 weeks as well as in age matched controls. Systolic arterial pressure and left ventricular weight normalized to body weight were each increased with either model of experimental hypertension and were normalized with high-dose spironolactone treatment. Myocardial fibrosis induced by chronic aldosterone administration was comparable to renovascular hypertension and occurred in the pressure overloaded, hypertrophied left and in the normotensive, nonhypertrophied right ventricle. The competitive aldosterone receptor antagonist, spironolactone, was able to prevent fibrosis in both ventricles in either model of arterial hypertension irrespective of the development of left ventricular hypertrophy and hypertension. To examine whether aldosterone stimulates collagen synthesis in adult rat cardiac fibroblasts collagen synthesis, normalized per total protein synthesis, was measured by 3H-proline incorporation in cultured fibroblasts after 24 hours incubation with aldosterone at 10(-11) to 10(-6) M concentrations, or with 10(-9) M aldosterone + 10(-9) M spironolactone. Under serum-free conditions, aldosterone was able to stimulate collagen synthesis in a dose-dependent manner and at concentrations (10(-9) M) which were comparable to stimulated states in vivo (e.g., renovascular hypertension, or chronic heart failure). At equimolar concentrations, spironolactone abolished the aldosterone-mediated increase in collagen synthesis. Thus, in-vivo and in-vitro evidence could be provided that the mineralocorticoid, aldosterone, plays a pivotal role in promoting myocardial fibrosis and that could be antagonized by its competitive receptor blocker, spironolactone. These cardioprotective effects of spironolactone may explain the prognostic value of anti-aldosterone therapy in patients with severe chronic heart failure evaluated in the RALES mortality trial.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.

The regulatory role of AT 1 receptor on activated HSCs in hepatic fibrogenesis:effects of RAS inhibitors on hepatic fibrosis induced by CCl(4).

AIM:To assess the effect of ACE inhibitor and Ang II type 1 (AT1) receptor antagonist in preventing hepatic fibrosis caused by CCl(4) administration in rats;to investigate whether or not there are expression of AT 1 receptors on hepatic stellate cells; and to observe the effect of Ang II on proliferation and ECM synthesis of cultured HSCs.METHODS:Studies were conducted in male Sprague-Dawley rats. Except for the hepatofibrotic model group and the control group, in three treated groups, either enalapril (5mg/kg), or losartan (10mg/kg), or enalapril + losartan were given to the fibrotic rats by daily gavage, and saline vehicle was given to model and normal control rats. After 6 weeks, liver fibrosis was assessed directly by hepatic morphometric analysis, which has been considered the gold standard for the quantification of fibrosis. The expressions of AT 1 receptors and (alpha-mooth muscle actin,alpha-SMA) in liver tissue or isolated hepatic stellate cells (HSCs) were detected by immunohistochemical techniques. The effect of Ang II on HSC proliferation was determined by MTT method. Effect of Ang II on collagen synthesis of HSCs was determined by (3)H-proline incorporation.RESULTS:Contrasted to the fibrosis in rats of the model group, groups of rats treated with either enalapril or losartan, or a combination of two drugs showed a limited expansion of the interstitium (4.23 plus minus 3.70 vs 11.22 plus minus 4.79, P<0.05), but no difference was observed among three treated groups (5.38 plus minus3.43, 4.96 plus minus 2.96, 4.23 plus minus 2.70, P>0.05). Expression of AT 1 receptors was found in fibrotic interstitium of fibrotic rats, whereas in normal control rats they were limited to vasculature only to a very slight degree. AT 1 receptors were also expressed on activated HSCs in the culture. At concentrations from 10(-9) to 10(-5)mol/L, Ang II stimulated HSC proliferation in culture in a dose dependent manner. Increasing Ang II concentrations produced corresponding increases in (3)H-proline incorporation. Differences among groups were significant.CONCLUSION:Angiotensin converting enzyme inhibitors and AT 1 blocker may slow the progression of hepatic fibrosis;activated HSCs express AT 1 receptors, and Ang II can stimulate the proliferation and collagen synthesis of HSCs in a dose-dependent manner; and activation of RAS may be related to hepatic fibrogenesis induced by CCl(4).

Fibroblast Differentiation in Subcutaneous Fibrosis after Postmastectomy Radiotherapy

In order to acquire a better understanding of the mechanism of radiation-induced fibrosis, we studied the differentiation of normal skin fibroblasts cultured from breast cancer radiotherapy patients with different risk of fibrosis. The differentiation state of fibroblasts was characterized in clonal cultures using established cytomorphological criteria. Collagen synthesis was determined by 3H-proline incorporation into pepsin-resistant protein. Radiation-induced inactivation of fibroblasts was paralleled by an increase in terminally differentiated fibrocytes, demonstrating that premature terminal differentiation is an important response to irradiation of fibroblasts from radiotherapy patients. Surviving colony-forming fibroblasts showed a change in differentiation with an increase in the ratio L : E of progenitor fibroblasts in late (L) compared to early (E) differentiation states. Furthermore, increased collagen production was observed after irradiation. The results provide evidence supporting a role of terminal fibroblast differentiation in radiation-induced fibrosis and imply that the progenitor population surviving radiotherapy might be more prone to terminal differentiation than before radiotherapy.

The effects of aging, antiinflammatory drugs, and ultrasound on the in vitro response of tendon tissue.

We investigated the effects of aging and various treatments on rat patellar tendon using an in vitro model. In the first part of the study, the 3H-thymidine and 3H-proline incorporation rates at 12 and 24 hours were determined in transected patellar tendon explants from young (21 days), intermediate age (8 to 10 weeks), and older (4 to 6 months) rats. In the second part, the same incorporation rates were measured in the older tendon explants in response to treatment with control medium, medium with a high and low concentration of indomethacin, and medium with a high and low concentration of dexamethasone. Finally, the effects of ultrasound treatment were measured and compared with a sham ultrasound treatment. The results indicated an age-dependent response of the tendon. The youngest specimens consistently showed the highest incorporation rates. The addition of a high concentration of dexamethasone resulted in a small negative effect on the 3H-thymidine incorporation. Ultrasound and indomethacin had no significant effects. This study indicates that aging is associated with a lower metabolic activity of tendon. In this model, currently used treatment methods failed to result in direct positive effects on tendon tissue, and a high concentration of dexamethasone appeared to have a small negative effect.

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared with L‐ascorbate in human intestinal smooth muscle cells

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of l-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 μM. By contrast, l-ascorbate was required at 4–5 times the concentration for the same response. However, at 20 μM, both palmitoyl and l-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas l-ascorbate had no effect. Palmitoyl ascorbate and l-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or l-ascorbate on the activity of a procollagen α2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than l-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro. J. Cell. Biochem. 73:312–320, 1999. © 1999 Wiley-Liss, Inc.

Palmitoyl ascorbate: Selective augmentation of procollagen mRNA expression compared withL-ascorbate in human intestinal smooth muscle cells

The effect of 6-O-palmitoyl ascorbate on procollagen mRNA levels, collagen synthesis, and collagen secretion was investigated and compared with the effect of L-ascorbate in human intestinal smooth muscle (HISM) cells in vitro. Collagen synthesis, determined by the incorporation of 3H-proline into pepsin-resistant, salt-precipitated collagen, increased in a concentration-dependent manner in response to palmitoyl ascorbate. There was a twofold increase in collagen synthesis at 2.5 and 5 microM. By contrast, L-ascorbate was required at 4-5 times the concentration for the same response. However, at 20 microM, both palmitoyl and L-ascorbate induced similar 2.7-fold increases in collagen synthesis. Palmitoyl ascorbate induced a 1.6- and 3.5-fold increase in steady-state levels of procollagen I and III mRNA levels respectively, whereas L-ascorbate had no effect. Palmitoyl ascorbate and L-ascorbate induced similar increases in the amounts of newly synthesized procollagen secreted into the medium and in the amounts of collagen types I, III and V accumulating in the cell layer. There was no effect of either palmitoyl ascorbate or L-ascorbate on the activity of a procollagen alpha2 (I) promoter construct transiently transfected into HISM cells. Palmitoyl ascorbate augments HISM cell procollagen synthesis and mRNA levels more efficiently than L-ascorbate. This property may be due to the greater resistance of the ascorbate ester to oxidation and suggests that palmitoyl ascorbate could be an important agent for studies of collagen synthesis in vitro.

Pentoxifylline inhibits PDGF-induced proliferation of and TGF-beta-stimulated collagen synthesis by vascular smooth muscle cells.

There is growing evidence that pentoxifylline (PTX) may have potential value as an antiproliferative and antifibrogenic agent. To assess whether this drug may be of use in the prevention of atherosclerosis or restenosis after angioplasty, we investigated the ability of PTX to inhibit proliferation and collagen synthesis in rat vascular smooth muscle cells (VSMCs) under both basal and platelet-derived growth factor (PDGF)- or transforming growth factor-beta (TGF-beta)- stimulated conditions. Intracellular cyclic AMP (cAMP) and cyclic GMP (cGMP) levels were measured in confluent cells using enzyme immunoassay kits. Cell proliferation was measured by methyltetrazolium assay. Cell cycle distribution was determined by flow cytometry. Total collagen synthesis was measured by 3H-proline incorporation assay. Expression of collagen alpha 1(I) and collagen alpha 1(III) mRNAs was detected by northern blotting. Addition of PTX to VSMC cultures suppressed both basal and PDGF-AB (25 ng/ml)-driven cell proliferation, in conjunction with a cell cycle blockade at the G1/S phase at 24 h. This effect was predominantly cAMP-dependent, as PTX increased cAMP in a dose-dependent manner (0.03 to 0.33 mg/ml) but not cGMP level, and the addition of dibutyryl-cAMP (0.2 to 2 m m) closely mimicked the effect of PTX. Furthermore, co-incubation with a selective inhibitor of cAMP-dependent protein kinase (PKA), H-89 (2.0 microm), or an N -myristoylated PKA pseudosubstrate nonapeptide, m-phi PKA (10 microm), prevented the antimitogenic effect of PTX. PTX also suppressed both basal and TGF- beta 1-augmented collagen alpha 1(I) and collagen alpha 1(III) mRNA levels beginning at 24 h, and attenuated both basal and TGF-beta 1 (5 ng/ml)-stimulated total collagen synthesis at 48 h. Co-incubation with H-89 or m-phi PKA reversed PTX-attenuated collagen alpha 1(I) and collagen alpha 1(III) mRNA levels at 24 h. These data suggest that the antimotigenic and anticollagen effects of PTX were mediated predominantly through a cAMP-PKA effector pathway. The dual effect of PTX on VSMC proliferation and collagen synthesis may form the rationale for animal or clinical trials for the treatment of vascular occlusion due to atherosclerosis and restenosis following angioplasty.

Phytoestrogens inhibit growth and MAP kinase activity in human aortic smooth muscle cells.

-Estrogens are known to induce cardioprotective effects by inhibiting smooth muscle cell (SMC) growth and neointima formation. However, the use of estrogens as cardioprotective agents is limited by carcinogenic effects in women and feminizing effects in men. If noncarcinogenic and nonfeminizing estrogenlike compounds, such as natural phytoestrogens, afford cardioprotection, this would provide a safe method for prevention of cardiovascular disease in both men and women. Therefore, we evaluated and compared in human aortic SMCs the effects of phytoestrogens (formononetin, genistein, biochanin A, daidzein, and equol) on 2.5% fetal calf serum-induced proliferation (3H-thymidine incorporation and cell number), collagen synthesis (3H-proline incorporation), and total protein synthesis (3H-leucine incorporation) and on PDGF-BB (25 ng/mL)-induced migration (modified Boydens chambers). Moreover, the effects of phytoestrogens on PDGF-BB (25 ng/mL)-induced mitogen-activated protein kinase (MAP kinase) activity in SMCs was also studied. Phytoestrogens inhibited proliferation, collagen and total protein synthesis, migration, and MAP kinase activity in a concentration-dependent manner and in the following order of potency: biochanin A>genistein>equol>daidzein>formononetin. In conclusion, our studies provide the first evidence that in human aortic SMCs phytoestrogens inhibit mitogen-induced proliferation, migration and extracellular matrix synthesis and inhibit/downregulate MAP kinase activity. Thus, phytoestrogens may confer protective effects on the cardiovascular system by inhibiting vascular remodeling and neointima formation and may be clinically useful as a safer substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Effect of in vitro Aging on the Modulation of Protein and Fibronectin Biosynthesis by the Elastin-Laminin Receptor in Human Skin Fibroblasts

The 67-kD elastin-laminin receptor (ELR) subunit which carries the recognition site for elastin peptides (EP) is a lectin. Its binding with galactosides can modulate the kinetics of its interaction with its ligand, EP. In this study the biosynthesis of proteins, collagen and fibronectin were evaluated in the presence of agonists and antagonists of the receptor on human skin fibroblasts. The biosynthesis of total proteins determined by ³H-proline incorporation and of fibronectin (by immunoprecipitation) were shown to increase with passage number. The presence of 1 µg/ml κ-elastin (EP) in the culture medium increased both total proteins and fibronectin biosynthesis. Melibiose, an agonist of the receptor at 5 µg/ml (140 µM), decreased both proteins and fibronectin biosynthesis in the culture medium of human skin fibroblast at the 10th and 15th passage. These results show that the ELR can control the biosynthetic mechanisms of some of the macromolecular constituents of extracellular matrix such as fibronectin and moderate its age and passage-dependent upregulation.

Circadian Rhythms in the Incorporation and Secretion of 3H-proline by Odontoblasts in Relation to Incremental Lines in Rat Dentin

Circadian incremental lines are universally found in the dentin of animals. They are believed to be caused by functional changes in odontoblasts over 24 hrs. However, the mechanism of rhythmic dentin formation has not yet been elucidated. In the present study, we investigated whether there is a 24-hour rhythm in the collagen-synthetic and secretory activities of odontoblasts by radioautography with 3H-proline as a tracer. Six different groups of rats were injected with 3H-proline at 0000, 0400, 0800, 1200, 1600, or 2000 after the animals had become acclimated to a 12/12-hour light-dark illumination cycle for 2 wks. One hour after the injection, the maxillary incisors were removed and processed for radioautographic study. The silver grains of 3H-proline were most intense over odontoblasts and predentin during the environmental light period, while the nadir occurred during the dark period. The peak value was approximately two-fold higher than the minimum value. Moreover, in the dentin from rats that had been infused with 3H-proline continuously for 10 days by means of osmotic minipumps, silver grains of 3H-proline were heavily distributed over the dark hematoxylin-stained incremental lines. Thus, we demonstrated that odontoblasts show circadian rhythm with regard to collagen synthesis and secretion. These rhythms in odontoblastic function may be responsible for circadian incremental lines in dentin.

Effects of ethanol on gene expression in rat bone: transient dose-dependent changes in mRNA levels for matrix proteins, skeletal growth factors, and cytokines are followed by reductions in bone formation.

Several studies were performed in female rats to determine dose and time course changes in mRNA levels for matrix proteins in bone after a single administration of ethanol. As expected, dose-dependent transient increases in blood ethanol were measured. Additionally, there was mild hypocalcemia with no change in immunoreactive parathyroid hormone. Coordinated dose-dependent increases in mRNA for type 1 collagen, osteonectin, and osteocalcin were noted in the proximal tibial metaphysis 6 hr after ethanol was given, with the peak values occurring at a dose of 1.2 g/kg (0.4 ml). Similar increases in mRNA levels for matrix proteins were noted in lumbar vertebrae after ethanol treatment. The changes were specific for bone; ethanol had no effect on mRNA levels for matrix proteins in the uterus or liver, although the mRNA concentrations tended to be reduced in uterus. Message levels for several cytokines implicated in the regulation of bone turnover were also assayed; mRNA levels for transforming growth factor-beta1, transforming growth factor-beta2, interferon-gamma, and interleukin-6 were unchanged at doses ranging from 0.14 to 1.7 g/kg. At the highest dose of ethanol, the mRNA level for tumor necrosis factor-alpha was elevated while the level for insulin-like growth factor-1 was reduced. The time course effects of ethanol (0.4 ml dose) were determined in a separate experiment. Ethanol resulted in a transient increase in mRNA levels for the three bone matrix proteins assayed. However, matrix protein synthesis, as determined by incorporation of 3H-proline into the proximal tibial metaphysis, was not changed after 6 hr. The changes in mRNA levels for the matrix proteins were preceded by brief, transient decreases in mRNA levels for interleukin-1beta, interferon-gamma, and migration inhibitory factor, and followed by a more prolonged decrease in the mRNA level for insulin-like growth factor-1. A subsequent study was performed to determine the effects of repetitive daily treatment with ethanol on rat bone. After 7 days, there were highly significant decreases in the mRNA level for type 1 collagen, as well as decreased bone formation. These results suggest that ethanol may alter bone metabolism by disturbing signal transduction pathways that regulate the expression of genes for bone matrix proteins, skeletal growth factors, and cytokines.

The role of reactive oxygen species in adriamycin and menadione-induced glomerular toxicity

Redox cycling leading to oxidative stress has been proposed as the mechanism by which adriamycin induces glomerular toxicity in rats. The present study compares the extent of the oxidative stress and cytotoxicity induced by adriamycin to menadione (a model redox cycling quinone) in freshly isolated rat glomeruli. Adriamycin and menadione (25 μM) decreased de novo protein synthesis (measured by 3H-proline incorporation into acid-precipitable glomerular protein) by 50 and 85%, respectively, in 2 h. By contrast, menadione at 25 μM reduce glomerular membrane integrity (as assessed by lactate dehydrogenase leakage), adriamycin reduced membrane integrity at 500 μM adriamycin. Reactive oxygen species (ROS) were measured by the oxidation of dihydrodichlorofluorescein. Menadione (25 μM) and adriamycin (25 μM) increased ROS formation to 260 and 156% of controls after 30 min incubation, respectively. Oxidative stress was assessed by measuring the intracellular level of reduced glutathione (GSH) and the decrease of the NADPH/NADP + ratio which stimulates the pentose phosphate pathway (PPP): (a) menadione (25–100 μM) reduced glomerular GSH to 10–20% of controls, adriamycin (25–100 μM) had no effect; (b) menadione (10 μM) increased PPP activity 6-fold, while adriamycin (125 μM) had only a 2-fold effect. Although adriamycin and menadione generate extensive ROS and decrease protein synthesis, there was no correlation between the extent of oxidative stress and cytotoxicity in glomeruli exposed to adriamycin. These results suggest that oxidative stress may not be the primary mechanisms by which adriamycin induces selective glomerular toxicity.

17Beta-estradiol, its metabolites, and progesterone inhibit cardiac fibroblast growth.

Postmenopausal women (PMW) have increased incidence of cardiovascular disease, and estrogen substitution therapy has been shown to have cardioprotective effects. Since abnormal growth of cardiac fibroblasts (CFs) is associated with hypertension and myocardial infarction and estrogen inhibits vascular smooth muscle cell (SMC) growth, it is feasible that estrogen may attenuate cardiac remodeling by inhibiting CF growth, and this possibility was investigated by using cultured CFs. 17Beta-estradiol and progesterone, but not 17alpha-estradiol, estrone, or estriol, inhibited 2.5% FCS-induced proliferation (DNA synthesis and cell number) and collagen synthesis (3H-proline incorporation) in a concentration-dependent manner and to a similar extent in male and female CFs. Compared to 17beta-estradiol, its metabolites 2-hydroxyestradiol and 2-methoxyestradiol were more potent in inhibiting FCS-induced DNA synthesis, collagen synthesis, and cell proliferation. The inhibitory effects of 17beta-estradiol and its metabolites were enhanced in presence of progesterone and 4-hydroxytamoxifen (high-affinity estrogen receptor ligand). Moreover, like estrogens, the dietary phytoestrogens biochanin A and daidzein inhibited FCS-induced growth of CFs. In conclusion, 17beta-estradiol, its metabolites, and progesterone inhibit CF growth in a gender-independent fashion. Moreover, hormone replacement therapy using 17beta-estradiol and progesterone may protect PMW against cardiovascular disease by inhibiting CF growth and cardiac remodeling; whereas estrogens that do not inhibit CF growth may be less effective in protecting PMW against cardiovascular disease. Finally, our studies provide evidence that phytoestrogens inhibit CF growth and may be clinically useful as a substitute for feminizing estrogens in preventing cardiovascular disease in both women and men.

Adenosine inhibits growth of human aortic smooth muscle cells via A2B receptors.

Adenosine inhibits rat vascular smooth muscle cell (SMC) growth. However, the effects of adenosine on human vascular SMC proliferation and synthesis of extracellular matrix proteins, such as collagen, are unknown. The objective of this study was to characterize the effects of exogenous and endogenous (SMC-derived) adenosine on human aortic SMC proliferation and collagen synthesis. Growth-arrested SMCs were stimulated with 2.5% fetal calf serum (FCS) in the presence and absence of adenosine, 2-chloroadenosine (stable adenosine analogue), and with agents that increase endogenous adenosine levels, including erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), dipyridamole, and iodotubericidin. All of these agents inhibited in a concentration-dependent manner FCS-induced SMC proliferation as assessed by DNA synthesis (3H-thymidine incorporation) and cell counting, as well as collagen synthesis (3H-proline incorporation). EHNA, dipyridamole, and iodotubericidin increased extracellular levels of adenosine by 1.7-fold to 18-fold when added separately to SMCs, and EHNA+iodotubericidin and EHNA+iodotubericidin+dipyridamole increased extracellular adenosine levels by more than 392-fold. Both KF17837 (selective A2 antagonist) and DPSPX (A1/A2 antagonist), but not DPCPX (selective A1 antagonist), blocked the antimitogenic effects of 2-chloroadenosine, EHNA, and dipyridamole on DNA and collagen synthesis, suggesting the involvement of A2A and/or A2B, but excluding the participation of A1, receptors. The lack of effect of CGS21680 (selective A2A agonist), excluded involvement of A2A receptors and suggested a major role for A2B receptors. A comparison of the inhibitory potencies of 2-chloroadenosine, N6-cyclopentyladenosine (selective A1 agonist), NECA (A1/A2 agonist), and MECA (A1/A2 agonist) were consistent with an A2B receptor subtype mediating the inhibitory effects of adenosine on human aortic SMC proliferation. In conclusion, human aortic SMCs synthesize adenosine, and exogenous as well as endogenous (SMC-derived) adenosine inhibits SMC proliferation and collagen synthesis via activation of A2B receptors.

Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-β 1

Failure to reepithelialize is the major clinical problem in venous ulcers. It is not clear whether the problem resides with keratinocytes or with inadequate and improper formation of extracellular matrix. In this study, we characterized the biosynthetic activity and response to transforming growth factor-β 1 (TGF-β) of dermal fibroblast cultures isolated from biopsies of venous ulcers and from normal thigh skin (controls) of seven patients. We found that baseline 3H-proline incorporation was similar in fibroblasts from venous ulcers and control skin ( p=0.1716). No difference was detected by ELISA between ulcer and control fibroblasts in the synthesis of total TGF-β ( p=0.2309), and the mRNA levels for α1(I) procollagen and TGF-β were comparable in both groups. However, TGF-β (0.1–5 ng/ml) enhanced collagen protein synthesis by more than 60% and in a dose-dependent manner ( r=0.997) in control fibroblast cultures, while failing to stimulate collagen production by venous ulcer fibroblasts ( p=0.0001). This unresponsiveness to TGF-β was associated with up to a fourfold decrease in TGF-β Type II receptors. We conclude that fibroblasts from the edge of non-healing venous ulcers are unresponsive to the action of TGF-β, and that this blunted response may cause faulty deposition of the extracellular matrix needed for reepithelialization and wound healing.

Interleukin-6 in Tear Fluid After Photorefractive Keratectomy and Its Effects on Keratocytes in Culture

To investigate the participation of interleukin-6 (IL-6) after photorefractive keratectomy (PRK) and its possible roles and sources in corneal wound healing. IL-6, levels were measured in the tear fluids of patients before and after PRK and in conditioned media of human corneal epithelial cells and keratocytes. Its effects on total collagen and collagen-type synthesis by keratocytes were studied with a 3H-proline incorporation assay and Northern blot analysis. Zymography was used to evaluate the metalloproteinase content in the conditioned medium of IL-6-stimulated keratocytes. IL-6 is present in the tear fluid samples after photorefractive keratectomy, possibly synthesized by epithelial cells and keratocytes. IL-6 stimulates collagen synthesis in general and collagen type I in particular. Furthermore, it reduces the production of MMP-2, the latent form of the metalloproteinase, by cultured keratocytes. The results suggest that IL-6 might be regarded as a mediator involved in corneal healing after excimer laser.

Transforming growth factor-β-induced collagen synthesis by human liver myofibroblasts is inhibited by α 2-macroglobulin

Background: Transforming growth factor-β (TGFβ) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFβ in different systems has been shown to be influenced by α 2-macroglubulin (α 2M), a serum protein with strong protease-scavenging and cytokine-binding properties. Aims: In the present study, α 2M derived from normal human plasma has been tested for its ability to modulate the TGFβ-induced collagen production by human liver fat-storing cells (FSC), which had transformed into α-smooth muscle actin-expressing myofibroblasts in culture. Methods: α 2M has been tested after activation with methylamine (α 2M-Me), an in vitro equivalent of protease activated α 2M. The binding of 125I-TGFβ1 to activated forms of α 2M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of 3H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of α 2M was studied by means of immunofluorescence. Results: TGFβ (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFβ. α 2M-Me reduced this TGFβ-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 μg/ml α 2M-Me onward. Upon addition of 100 μg/ml α 2M-Me the effect of TGFβ was reduced by 60% to 128±31% (mean±SD) of control values in the absence of TGFβ. Human liver myofibroblasts endocytosed α 2M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFβ-activity by α 2M-Me may be explained by receptor-mediated clearance of α 2M-TGFβ complexes by the cells. Conclusions: TGFβ-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated α 2M. From these results, we hypothesize that α 2M may have an antifibrogenic effect in vivo by interference with TGFβ-induced matrix synthesis during liver fibrosis.

Interleukin-1-producing plasma cells in close contact with hepatocytes in patients with chronic active hepatitis

Background/Aims: In chronic active liver diseases (CALD) with viral aetiology, a population of plasma cells localised in the piecemeal necrosis areas was previously detected by means of autoradiography after in vitro3H-proline incorporation, a method which proved much more sensitive than conventional immunohisto-chemical procedures. These plasma cells, characteristically located in niches among hepatocytes, in close contact with collagen fibrils, have been hypothesised to exert a role in fibrogenesis stimulation, and particularly in collagen synthesis, possibly through secretion of lymphokines. Specifically, we investigated the presence of interleukin-1, well known to play a crucial role in inflammation and production of collagen by epithelial cells, and to be present in activated plasma cells of myeloma.Methods: The immunohistochemical localisation of interleukin-1β in biopsies of patients suffering from chronic active hepatitis was studied, using an affinity-purified rabbit polyclonal antibody.Results: The strongest interleuki-1β immunostaining was observed in the above-described plasma cell population, identified by anti-immunoglobulin antibodies, and 3H-proline incorporation.Conclusions: The ability of plasma cells to produce interleukin-1 during viral CALD suggests that in these pathologies plasma cells play a major role, mainly of paracrine nature. Interleukin-1, possibly together with other mediators, might in turn stimulate the production of collagen. Hepatocytes of the piecemeal necrosis area appear to be possible candidates for this synthesis, as they show a significant labelling after 3H-proline incorporation, which is absent from hepatocytes far from necrotic areas.

Regulation and role of myocardial collagen matrix remodeling in hypertensive heart disease.

In hypertensive heart disease, reactive myocardial fibrosis represents as an excessive accumulation of fibrillar collagen within the normal connective tissue structures of the myocardium. The fact, that the myocardium of both ventricles is involved, irrespective of ventricular loading conditions, suggests that circulating factors, and not the hemodynamic load are primary responsible for this adverse response of the myocardial fibrous tissue. In various experimental in vivo models, it has been shown that myocardial fibrosis is always associated with activation of circulating or local renin-angiotensin-aldosterone systems (RAAS). Cardiac collagen metabolism is regulated by cardiac fibroblasts which express mRNAs for types I and III collagens, the major fibrillar collagens in the heart, and for interstitial collagenase or matrix metalloproteinase (MMP) 1 which is the key enzyme for interstitial collagen degradation. In order to elucidate the role of the RAAS effector hormones, angiotensin II (AngII) and aldosterone (ALDO), in the regulation of collagen synthesis or inhibition of MMP 1 production, adult human cardiac fibroblasts were cultured. Collagen synthesis was determined by 3H-proline incorporation, and MMP 1 activity by degradation of 14C-collagen measured under serum-free conditions in confluent fibroblasts after 24 hour-incubation with either AngII or ALDO over a wide range of concentrations (10(-11)-10(-6)M). In addition, the effects of the mineralocorticoid, deoxycorticosterone (DOC), and prostaglandin E2 (PGE2) on cardiac fibroblast function were determined. Compared with untreated control fibroblasts, collagen synthesis, normalized per total protein synthesis, showed a significant and dose-dependent increase after incubation with either mineralocorticoid hormone, ALDO or DOC, or after incubation with AngII. In contrast, collagen synthesis of cardiac fibroblasts was significantly decreased by PGE2 treatment. AngII type 1 or mineralocorticoid receptor antagonists, respectively, were able to completely inhibit the AngII- or mineralocorticoid-mediated increase of collagen synthesis. Furthermore, AngII significantly decreased MMP 1 activity while ALDO or DOC had no effect on cardiac fibroblast-mediated collagen degradation. In contrast, PGE2 significantly increased MMP 1 activity. Thus cardiac fibroblast function is modulated by either effector hormone of the RAAS, AngII and ALDO, via specific receptors that lead to progressive myocardial fibrosis in disease states where circulating or local RAAS is activated, i.e., in hypertensive heart disease. In contrast, PGE2, which would be elevated in myocardial tissue after angiotensin-converting enzyme inhibition, counteracts the fibrotic effects of the RAAS on myocardial tissue.