Differential response to transforming growth factors-β1 and β2: connective tissue deposition in animal models

The ability of transforming growth factor (TGF)-beta(1) and TGF-beta(2) to promote connective tissue deposition were compared in different animal models. A single subcutaneous injection of TGF-beta(2) in collagen/heparin gel carrier promoted markedly more extensive development of connective tissue than TGF-beta(1) at the site of injection in both neonatal and adult mice. Both TGF-beta(1) and TGF-beta(2) promoted deposition of dense and well-vascularized connective tissue matrix infiltrated with macrophages and fibroblasts. However, the results of immunohistochemical analyses suggested that TGF-beta(2) promoted an accumulation of more macrophages in the connective tissue than TGF-beta(1). Similar differences in the extent of connective tissue development were observed in neonatal mice when these factors were administered as a solution, without the collagen/heparin gel carrier. TGF-beta(2) was also more potent than TGF-beta(1) in domestic pigs. However, in guinea pigs, TGF-beta(1) promoted more extensive connective tissue development than TGF-beta(2). These results suggest that the differential connective tissue response to TGF-beta(1) and TGF-beta(2) is species dependent. However, the differences in the physical and chemical properties of these factors may account in part for the differential response as well.     

β2-Microglobulin and Low-Flux SyntheticDialyzers

Abstract: The aim of this study was to compare the effect on β₂-microglobulin (β₂-M) plasma levels of dialyzers with 3 low-flux synthetic membranes and regenerated cellulose (Cuprophan) in 12 chronic dialysis patients. The synthetic membrane materials chosen were low-flux polymethylmethacrylate (PMMA), low-flux polysulfone (PS 400), and polycarbonate-polyether (Gambrane). Adequate and comparable removal of small solutes was provided by dialyzers with all 4 membrane materials used under similar conditions. A significant reduction of β₂-M plasma levels was seen only with Gambrane while the other 2 synthetic membrane materials gave rise to increases similar to those known to occur with Cuprophan. After correction for the hemoconcentration caused by ultrafiltration, dialysis with Gambrane showed a 24% lower plasma β₂-M level while the β₂-M concentrations with the other 3 membrane materials were practically unchanged. In addition, the efficiency of Gambrane dialyzers in β₂-M removal was able to significantly lower the predialysis plasma β₂-M levels after only 5 dialysis sessions. The hemocompatibility of the 3 synthetic low-flux membranes as judged by the white blood cell (WBC) count and complement activation was similar and therefore cannot be used to explain the different β₂-M plasma levels. In anticipation of gaining further insight into the mechanisms of accumulation and deposition of β₂-M in dialysis patients, a worthwhile approach may be to use a low-flux membrane such as Gambrane which combines removal with protection against potential activating factors in the dialysis fluid.     

Characterization of a murine monoclonal antibodv specific for swine β1 integrin

Abstract: Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160–1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM 160–1 A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human β₁ integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine α₂ integrin. Therefore, the NaM 160–1 A3 antibody was directed against porcine β₁ integrin and allowed the purification of the complex α₂β₁ also termed Very Late Antigen 2 (VLA-2). It did not recognize human β₁ integrin.     

Glomerular TGF-β1 expression in children with nephrotic syndrome

Summary: Transforming growth factor-β (TGF-β) has been suggested to contribute to the onset and/or progression of glomerulonephritis. However, data of TGF-β in connection with the pathogenesis of nephrotic syndrome are still insufficient. an immunohistochemical study on the glomerular TGF-β₁ in nephrotic syndrome was carried out in order to clarify the pathological role of this substance. Ten cases of nephrotic syndrome were subdivided histologically into two groups: (i) six cases of minor glomerular abnormality (group M); and (ii) four cases of focal glomerular sclerosis (group F). Two cases with normal glomeruli, one case of normal portion of adult renal cell carcinoma, one case of normal portion of Wilm's tumour were also studied as controls. Four cases of asymptomatic haematuria (group H) and four cases of chronic non-IgA glomerulonephritis syndrome (group C) were chosen compared to the nephrotic syndrome. Staining was evaluated semiquantitatively by light microscopy and by measuring the staining ratio compared to the glomerular area on an image analyzer. Both methods showed significantly larger staining in the glomeruli of group F, compared to group M. We concluded that TGF-β₁ plays an important role in the progression of nephrotic syndrome.     

Effects of hypotensive treatment with α2-agonist and β1-antagonist on cerebral haemodynamics in severely head injured patients

Therapy of post-traumatic brain oedema often includes preservation of high arterial blood pressure to avoid secondary ischaemic injuries to the brain. This practice can be questioned since high arterial blood pressure may aggravate brain oedema through raised hydrostatic capillary pressure, causing fluid filtration across the damaged blood-brain barrier. This latter view is in agreement with our clinical experience and therefore hypotensive therapy with an α₂-adrenergic agonist (clonidine) and a β₁-adrenergic antagonist (metoprolol) has become part of our treatment protocol for severely head injured patients to decrease the post-traumatic brain oedema. The present study is an attempt to analyse whether there are any direct local cerebrovascular effects of the hypotensive agents used, which also might influence intracranial pressure. Severely head injured patients were investigated. Heart rate, mean arterial blood pressure, intracranial pressure, cerebral blood flow and arterio-venous difference in oxygen content were measured before and after a bolus dose of clonidine (six patients) and metoprolol (nine patients).
Clonidine decreased mean arterial blood pressure and cerebrovascular resistance without affecting other parameters measured. Metoprolol decreased heart rate and mean arterial pressure, but had no effect on the cerebrovascular parameters.
The results show that clonidine and metoprolol have no, or only minor, direct influence on local cerebral haemodynamics in severely brain injured patients. This implies that if there is an intracranial pressure reducing effect of these drugs, as suggested, this must be due to other mechanisms, namely a reduction in capillary hydrostatic pressure secondary to decreased arterial blood pressure and heart rate.     

α1-Adrenoceptor pharmacome: α1L-Adrenoceptor and α1A-adrenoceptor in the lower urinary tract

α₁‐Adrenoceptors are involved in physiological functions such as urinary excretion and ejaculation in the lower urinary tract (LUT). Several α₁ antagonists are clinically used for the treatment of urinary obstruction in patients with benign prostatic hyperplasia. At present, three classical α₁‐adrenoceptor subtypes (α_1A, α_1B, and α_1D) have been identified, among which the α_1A and α_1D‐adrenoceptor subtypes have been regarded as the main targets of α₁ antagonist therapy for LUT symptoms. Prazosin has been used as a prototypic, classical antagonist, to characterize α₁‐adrenoceptors pharmacologically, (i.e. all classical α₁‐adrenoceptor subtypes show high‐affinity for the drug). However, we found that α₁‐adrenoceptors in the LUT show atypical low‐affinity for prazosin. Therefore, the concept α_1L‐receptor, which indicates α₁‐adrenoceptor(s) showing low‐affinity for prazosin has been introduced. A recent study demonstrated that the α_1L‐adrenoceptor is a specific phenotype present in the many intact tissues including human LUT, and that it originates from the ADRA1A gene. Therefore, the α_1L‐adrenoceptor in the LUT is now re‐defined as α_1A(L)‐adrenoceptor. The physiological and pharmacological difference between classical α_1A(H), and α_1A(L) which is the native receptor expressed in the LUT is of special interest as it provides fundamental bases for urological α_1A‐adrenoceptor blocking pharmacotherapy. Here, we briefly review the α₁‐adrenoceptors in the LUT with special reference to phenotype‐based (pharmacome) analysis.     

Exogenous transforming growth factor-β2 enhances connective tissue formation in transforming growth factor-β1—deficient, healing-impaired dermal wounds in mice

Connective tissue formation is markedly reduced in full-thickness mouse dermal wounds that are covered with synthetic, adherent, moisture vapor-permeable membrane when compared with formation in similar but nonoccluded wounds. The transforming growth factors-beta (TGF-beta) are a family of multifunctional peptides thought to have a critical role in the regulation of development and tissue repair. Treatment with exogenous TGF-beta(1) stimulated connective tissue formation in wounds covered with synthetic, adherent, moisture vapor-permeable membrane but had no effect on air-exposed wounds, suggesting that the quantity of endogenous TGF-beta(1) in wounds covered with synthetic, adherent, moisture vapor-permeable membrane was less than that in air-exposed wounds. Immunolocalization studies with an anti-TGF-beta(1) antibody confirmed that wounds covered with synthetic, adherent, moisture vapor-permeable membrane demonstrated markedly reduced levels of endogenous extracellular, matrix-associated TGF-beta(1) as early as 24 hours after wounding. Immunoreactive TGF-beta(2) was not detected. These findings suggest that endogenous TGF-beta(1), but not TGF-beta(2), is required for normal connective tissue formation in this model and that impaired healing is associated with low levels of TGF-beta(1). Histologic analysis confirmed previous demonstrations that exogenous TGF-beta(2) stimulates enhanced cellularity and connective tissue formation. Immunolocalization showed that exogenous TGF-beta(2) stimulates increased expression of endogenous TGF-beta(1). Northern blot analysis revealed that TGF-beta(2) increased the expression of genes encoding the alpha(1)-chain of types I and III collagens and tissue inhibitor of metalloproteinase-1. These observations show that TGF-beta(2) acts through a variety of mechanisms to stimulate repair in healing-impaired wounds that are also deficient in endogenous TGF-beta(1), but they do not distinguish between direct effects and indirect effects mediated by induced TGF-beta(1).     

Histochemical Studies of Testicular δ5 -3β-Hydroxysteroid Dehydrogenase and 17β-Hydroxysteroid Dehydrogenase after Chronic Indomethacin Administration in Rats Pretreated with Clomiphene-Citrate

Histochemical studies of testicular delta5-3beta-Hydroxysteroid Dehydrogenase and 17beta-Hydroxysteroid Dehydrogenase in sexually immature rats treated chronically with simultaneous Indomethacin and Clomiphene revealed greater inhibition in the enzyme activities when compared to Clomiphene treated animals alone. This suggests prostaglandin-inhibitors may be directly inhibitory to NAD-requiring enzymes involved in testicular steroid biosynthesis.     

Transforming growth factor-β3 affects plasminogen activator inhibitor-1 expression in fetal mice and modulates fibroblast-mediated collagen gel contraction

For over two decades, the precise role of transforming growth factor-beta (TGF-beta) isoforms in scarless healing of mammalian fetal skin wounds has generated much interest. Although their exact role remains to be established, it has been suggested that high TGF-beta3 activity may correlate with a scarless phenotype. Previously, we showed that plasminogen activator inhibitor-1 (PAI-1), a known TGF-beta downstream molecule and marker of fibrosis, is also developmentally regulated during fetal skin development. In this study, the relationship between TGF-beta3 and PAI-1 was investigated using embryonic day 14.5 TGF-beta3 knockout (ko) mice. The results showed increased PAI-1 expression in the epidermis and dermis of ko mice, using an ex vivo limb-wounding study. Furthermore, increased PAI-1 expression and activity was seen in embryo extracts and conditioned media of ko dermal fibroblasts. When TGF-beta3 knockout fibroblasts were placed into three-dimensional collagen matrices, they were found to have decreased collagen gel contraction, suggesting altered cell-matrix interaction. These findings provide a further avenue for the interactive role of TGF-beta3 and PAI-1 during fetal scarless repair.     

α1-Adrenoceptor subtypes and lower urinary tract symptoms

Abstract:   Benign prostatic hyperplasia (BPH) is a common cause of urinary outflow obstruction in aging men leading to lower urinary tract symptoms (LUTS). α₁-Adrenoceptors (α₁ARs) antagonists (blockers) have become a mainstay of LUTS treatment because they relax prostate smooth muscle and decrease urethral resistance, as well as relieving bladder LUTS symptoms. A review of key recent clinical trials suggests new insights into the role of specific α₁AR subtypes in the treatment of LUTS.     

Autocrine transforming growth factor-β1 activity and glycosaminoglycan synthesis by human cutaneous scar fibroblasts

Transforming growth factor-beta(1) is a well-known and potent biological response modifier that plays an important role in tissue repair and fibrosis. Among the extracellular constituents known to accumulate in fibrotic tissues, glycosaminoglycans are prominent. In this study we examined transforming growth factor-beta(1) synthesis by human dermal fibroblasts derived from both normal and fibrotic cutaneous tissues. We studied the influence of transforming growth factor-beta(1) on glycosaminoglycan synthesis and explored the role of transforming growth factor-beta(1) as an autocrine mediator of its own expression. These investigations are directed at understanding the persistence of the fibrotic phenotype in scarred skin. Transforming growth factor-beta(1) activity was measured by means of a mink lung epithelium growth inhibitory assay. Replicate explants (n = 3) of fibroblasts each derived from normal skin, normal scar, or hypertrophic scar were studied by adding exogenous transforming growth factor-beta(1) at a concentration range of 0 to 10 ng/ml. The resulting conditioned media were removed and assayed for transforming growth factor-beta(1) activity, then the cells were pulsed for an additional 24 hours with radiolabeled glycosaminoglycan precursor, [(3)H]-glucosamine, to evaluate glycosaminoglycan production. Cell-free glycosaminoglycan synthetic profiles were also developed. Transforming growth factor-beta(1) was found to cause a dose-dependent increase in glycosaminoglycan synthesis in hypertrophic scar and normal skin but not in normal scar fibroblasts in cell-mediated glycosaminoglycan synthesis; the reverse was observed in cell-free glycosaminoglycan synthesis, where transforming growth factor-beta(1) increased glycosaminoglycan synthesis in normal scar but not in normal skin or hypertrophic scar. Most endogenous transforming growth factor-beta(1) existed in latent form for normal skin cells but in active form for normal scar and hypertrophic scar fibroblasts.     

Influence of Blood Flow on Adsorption of β2-Microglobulin onto AN69 Membrane

Abstract: Adsorption onto the dialyzer membrane is a contributing factor to the elimination of β₂-microglobulin (β₂M) from the sera of uremic patients. The purpose of this prospective study was to ascertain the influence of the blood flow rate on adsorption of β₂M onto the polyacrylonitrile (AN69) hollow-fiber dialyzer membrane in 8 pa tients during regular hemodialysis (HD). Blood first passed through a low-flux polysulfone dialyzer and then through an AN69 dialyzer, which was not in contact with the dialysis fluid. During the investigation period (first hour of the HD session), the blood flow rate was 100 ml/min (first part of the study), 200 mumin (second part of the study), and 300 ml/min (third part of the study). Ultrafiltration was not performed during the investigation period. At the start of the HD sessions, the serum concentration of β₂M in the afferent blood line did not differ significantly among the 3 parts of the study. Serum β₂M was measured in samples taken from the afferent and efferent blood lines of the AN69 dialyzer at 5,10, 15, 30, 45, and 60 min. The serum β₂M concentration decreased significantly in blood that had passed through the AN69 dialyzer. This decrease, indicating membrane adsorption, was maximal during the first part and minimal during the third part of study. The decrease in the contact time between the blood and the AN69 could be the underlying cause. The calculated quantities of β₂M adsorbed onto the AN69 membrane (44.2 ± 10.2, 43.2 ± 12.1, and 42.6 ± 17.3 mg) did not differ significantly among the 3 parts of the study. These results suggest that an increase in blood flow rate from 100 to 300 ml/min did not significantly affect the quantity of β₂M adsorbed onto the AN69 membrane.     

Role of β2 integrins in glomerular leucocytes in Henoch-Schoenlein purpura nephritis: an age-matched comparative study with immunoglobulin A nephropathy

SUMMARY: A comparative immunohistological study was performed for the glomerular deposition of complements (C1q and C3c), fibrin/fibrinogen‐related antigen (FRA), the expression of intercellular adhesion molecule‐1 (ICAM‐1), and the infiltration of leucocytes bearing β₂ integrins (leucocyte function associated antigen‐1 (LFA‐1), complement receptor 3 (CR3) and complement receptor 4 (CR4)) on renal biopsy specimens from 49 cases with Henoch‐Schoenlein purpura nephritis (HSPN), and 49 age‐matched cases with immunoglobulin A nephropathy (IgAN). the glomerular expression of ICAM‐1 was signifcantly correlated with the glomerular infiltration of leucocyte function associated antigen (LFA)‐1⁺ leucocytes in both diseases, and with that of CR3⁺ leucocytes in HSPN. the expression of ICAM‐1 was closely localized with the infiltration of LFA‐1⁺ leucocytes in the study with double immunostaining. the incidence and intensity of glomerular deposition of FRA were significantly higher in HSPN than in IgAN (P< 0.001), and those of C3c were significantly lower in HSPN than in IgAN (P< 0.001). the glomerular deposition of FRA was significantly correlated with the glomerular infiltration of CR4⁺ leucocytes in HSPN (P<0.05) but not in IgAN. In contrast, the glomerular deposition of C3c was significantly correlated with the glomerular infiltration of CR4⁺ leucocytes in IgAN (P<0.05), but not in HSPN. Studies with double immunostaining revealed a close association of CR4⁺ leucocytes with FRA deposition in HSPN and with C3c deposition in IgAN, respectively. the number of glomerular leucocytes bearing β₂ integrins was significantly correlated with urinary protein at the time of renal biopsy in both diseases. These results suggested the differential roles of β₂ integrins in the induction of glomerular injury in HSPN and IgAN. the ICAM‐1/LFA‐1 interaction may commonly be involved in the glomerular infiltration of leucocytes in both diseases. the ICAM‐1/CR3 interaction may be involved only in HSPN. Complement receptor 4 may function as a fibrin/fibrinogen receptor in HSPN, while CR4 may function as a complement receptor in IgAN.     

End-stage renal failure in New Zealand Maori: an analysis of circulating transforming growth factor-β1

SUMMARY: There is a high incidence of end-stage renal disease in New Zealand Maori. Reasons for this have not been established. Transforming growth factor-β, (TGF-β₁) is a profibrogenic cytokine, which stimulates the secretion of extracellular matrix components, and has been implicated in the pathogenesis of kidney failure. the aim of this study was to examine TGF-β₁ in the serum of haemodialysis patients at our institution, in order to determine whether there was an upregulation of TGF-β₁ in Maori. A TGF-Prspecific sandwich enzyme-linked immunosorbant assay was used to measure active TGF-β from the sera of 74 haemodialysis patients, and 19 healthy Maori without renal disease, diabetes or hypertension. In addition, clinical and laboratory markers were examined in the haemodialysis patients studied. There was no association between TGF-β₁ and ethnicity in the groups studied. Transforming growth factor-β₁ protein appeared to be inversely related to age. but was not associated with parameters of survival on dialysis such as serum albumin or measures of dialysis adequacy. Although there was a significantly higher incidence of type II diabetes mellitus in the Maori ( P < 0.001) in comparison to European patients, the glycaemic control was comparable between the groups, as were all other laboratory and clinical parameters studied. This is the first study to examine the fibrogenic growth factor TGF-β₁ in New Zealand Maori. Thus, an endogenous increase in TGF-β₁ in Maori does not appear to be implicated in the increased incidence of end-stage renal disease in this population.     

Kinetics of 131I-β2 Microglobulin in Hemodialysis Patients: Assessment Using Total Body Counting

Abstract: The kinetics of ¹³¹I-β₂-microglobulin (β₂-M) were studied using external total body gamma counting in a low noise chamber after administration of trace doses of radioactivity (4 μCi) in 14 uremic patients treated by either hemodialysis or hemofiltration. Data were collected over a 1 week period that included 3 dialysis sessions. The following artificial membranes were used: Cuprophan, polyacrylonitrile AN69, polysulfone, polymethylmethacrylate (PMMA), and polyamide. Radiolabeled β₂-M excretion by an extrarenal route was nearly nonexistent. The ¹³¹I-β₂-M half-life was between 2.4 and 8 days, shorter in patients with residual diuresis. A mean removal of 153 ± 33 mg/L of β₂-M was obtained per dialysis session with a highly permeable membrane. A hemofiltration session (25 L exchange per session) was slightly more efficient in removing β₂-M than a 4 h hemodialysis session with the same AN69 highly permeable membrane. The amounts of ¹³¹I-β₂-M binding on the membranes, expressed as β₂-M equivalents, were 0, 16, 54, 58, and 59 mg/m² for Cuprophan, polysulfone, polyacrylonitrile AN69, polyamide, and PMMA, respectively. In conclusion, the decrease of total body gamma counting directly reflected the β₂-M breakdown and removal in hemodialysis patients. Intact β₂-M was removed by convection with synthetic, highly permeable membranes. In addition, membrane adsorption accounted for 15% (polysulfone) to near 100% (PMMA) of the β₂-M removal per session. Adsorption was of the same magnitude regardless of the dialysis technique in use, indicating a membrane saturability process. None of the currently available dialysis procedures based on a 3 sessions per week schedule can balance β₂-M generation.