The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo.

OBJECTIVES: The aim of this study was to investigate the effect of hyperglycaemia on oxidative stress markers and inflammatory and matrix gene expression within tendons of normal and diabetic rats and to give insights into the processes involved in tendinopathy. METHODS: Using tenocytes from normal Sprague-Dawley rats, cultured both in control and high glucose conditions, reactive oxygen species (ROS) production, cell proliferation, messenger RNA (mRNA) expression of NADPH oxidase (NOX) 1 and 4, interleukin-6 (IL-6), matrix metalloproteinase (MMP)-2, tissue inhibitors of matrix metalloproteinase (TIMP)-1 and -2 and type I and III collagens were determined after 48 and 72 hours in vitro. In an in vivo study, using diabetic rats and controls, NOX1 and 4 expressions in Achilles tendon were also determined. RESULTS: In tenocyte cultures grown under high glucose conditions, gene expressions of NOX1, MMP-2, TIMP-1 and -2 after 48 and 72 hours, NOX4 after 48 hours and IL-6, type III collagen and TIMP-2 after 72 hours were significantly higher than those in control cultures grown under control glucose conditions. Type I collagen expression was significantly lower after 72 hours. ROS accumulation was significantly higher after 48 hours, and cell proliferation after 48 and 72 hours was significantly lower in high glucose than in control glucose conditions. In the diabetic rat model, NOX1 expression within the Achilles tendon was also significantly increased. CONCLUSION: This study suggests that high glucose conditions upregulate the expression of mRNA for NOX1 and IL-6 and the production of ROS. Moreover, high glucose conditions induce an abnormal tendon matrix expression pattern of type I collagen and a decrease in the proliferation of rat tenocytes.Cite this article: Y. Ueda, A. Inui, Y. Mifune, R. Sakata, T. Muto, Y. Harada, F. Takase, T. Kataoka, T. Kokubu, R. Kuroda. The effects of high glucose condition on rat tenocytes in vitro and rat Achilles tendon in vivo. Bone Joint Res 2018;7:362-372. DOI: 10.1302/2046-3758.75.BJR-2017-0126.R2.

Repair of osteochondral defects with a new porous synthetic polymer scaffold.

We developed a new porous scaffold made from a synthetic polymer, poly(DL-lactide-co-glycolide) (PLG), and evaluated its use in the repair of cartilage. Osteochondral defects made on the femoral trochlear of rabbits were treated by transplantation of the PLG scaffold, examined histologically and compared with an untreated control group. Fibrous tissue was initially organised in an arcade array with poor cellularity at the articular surface of the scaffold. The tissue regenerated to cartilage at the articular surface. In the subchondral area, new bone formed and the scaffold was absorbed. The histological scores were significantly higher in the defects treated by the scaffold than in the control group (p<0.05). Our findings suggest that in an animal model the new porous PLG scaffold is effective for repairing full-thickness osteochondral defects without cultured cells and growth factors.

Effects of platelet-rich plasma and triamcinolone acetonide on interleukin-1ß-stimulated human rotator cuff-derived cells.

OBJECTIVES: Triamcinolone acetonide (TA) is widely used for the treatment of rotator cuff injury because of its anti-inflammatory properties. However, TA can also produce deleterious effects such as tendon degeneration or rupture. These harmful effects could be prevented by the addition of platelet-rich plasma (PRP), however, the anti-inflammatory and anti-degenerative effects of the combined use of TA and PRP have not yet been made clear. The objective of this study was to determine how the combination of TA and PRP might influence the inflammation and degeneration of the rotator cuff by examining rotator cuff-derived cells induced by interleukin (IL)-1ß. METHODS: Rotator cuff-derived cells were seeded under inflammatory stimulation conditions (with serum-free medium with 1 ng/ml IL-1ß for three hours), and then cultured in different media: serum-free (control group), serum-free + TA (0.1mg/ml) (TA group), serum-free + 10% PRP (PRP group), and serum-free + TA (0.1mg/ml) + 10% PRP (TA+PRP group). Cell morphology, cell viability, and expression of inflammatory and degenerative mediators were assessed. RESULTS: Exposure to TA significantly decreased cell viability and changed the cell morphology; these effects were prevented by the simultaneous administration of PRP. Compared with the control group, expression levels of inflammatory genes and reactive oxygen species production were reduced in the TA, PRP, and TA+PRP groups. PRP significantly decreased the expression levels of degenerative marker genes. CONCLUSIONS: The combination of TA plus PRP exerts anti-inflammatory and anti-degenerative effects on rotator cuff-derived cells stimulated by IL-1ß. This combination has the potential to relieve the symptoms of rotator cuff injury.Cite this article: T. Muto, T. Kokubu, Y. Mifune, A. Inui, R. Sakata, Y. Harada, F. Takase, M. Kurosaka. Effects of platelet-rich plasma and triamcinolone acetonide on interleukin-1ß-stimulated human rotator cuff-derived cells. Bone Joint Res 2016;5:602-609. DOI: 10.1302/2046-3758.512.2000582.

Solubilization of angiotensin I-coverting enzyme from rabbit lung using trypsin treatment.

The solubilization of angiotensin I-converting enzyme (peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was carried out using trypsin treatment. A good recovery of 76% was obtained. The enzyme from solubilized fraction was purified using colums of Sephadex G-200, hydroxyapatite and DEAE-cellulose. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 24.3 units/mg protein for hippurylhistidylleucyl hydroxide and 0.182 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment for 5 h could be divided into two components: (i) an enzyme of molecular weight 300 000 (peak II) and (ii) an enzyme of molecular weight 145 000 (peak III), by Sephadex G-200 gel filtration. The molecular weight of the denatured enzyme was found to be 155 000 by disc gel electrophoresis in the presence of sodium dodecyl sulfate. Km values of peak II and peak III fraction for Hippuryl-His Leu-OH were 2.6 mM.

Affinity chromatography of angiotensin I-converting enzyme from rabbit lung using hippurylhistidylleucyl-OH.

Approximately 50-fold purification of angiotensin I-converting enzyme (Peptidyldipeptide hydrolase, EC 3.4.15.1) from rabbit lung was achieved by affinity chromatography using the synthetic substrate Hippuryl-His-Leu-OH. The specific activity of the enzyme was increased from 0.044 units/mg protein to 1.911 units/mg protein for Hippuryl-His-Leu-OH and from 0.33 nmol/min per mg protein to 13.8 nmol/min per mg protein for angiotensin I.

Purification of angiotensin I-converting enzyme from human lung.

Angiotensin I-converting enzyme (peptidyl dipeptide hydrolase, EC 3.4.15.1) was solubilized from the membrane fraction of human lung using trypsin treatment and purfied using columns of DE 52-cellulose, hydroxyapatite and Sephadex G-200. The purified enzyme was shown to convert angiotensin I to angiotensin II and also to inactivate bradykinin. The specific activity of the enzyme was 9.5 units/mg protein for Hippuryl-His-Leu-OH and 0.665 mumol/min per mg protein for angiotensin I. The enzymic activity obtained after trypsin treatment (1 mg/200 mg protein) for 2 h could be divided into three components: (i) an enzyme of molecular weight 290 000 (peak I), (ii) an enzyme of molecular weight 180 000 (peak II) and (iii) an enzyme of molecular weight 98 000 (peak III), by columns of DE 52-cellulose and Sephadex G-200. Km values of peak I, II and III fraction for Hippuryl-His-Leu-OH were identical at 1.1 mM. pH optimum of the enzyme was 8.3 for Hippuryl-His-Leu-OH.

Orthostatic hypertension with nephroptosis and aortitis disease.

A 53-year-old woman with nephroptosis and aortitis disease was found also to have orthostatic hypertension. When standing, she had high renin levels and normal catecholamine values, with a reduced baroreflex sensitivity. This orthostatic hypertension largely may be due to an activation of the renin system caused by nephroptosis and partly due to a reduced baroreflex sensitivity caused by aortitis. Captopril and propranolol hydrochloride were effective for the treatment of hypertension.

No evidence for the existence of a species-specific renin substrate in plasma from normal and bilaterally nephrectomized rats.

Renin substrate in plasma from normal and biolaterally nephrectomized rats was measured using an excess of rat or rabbit renin which had the same pressor activity when directly assayed in the rat. The amounts of angiotensin I generated with an excess of rat renin were similar to those generated with an excess of rabbit renin in plasma from normal and bilaterally nephrectomized rats. Further addition of an excess of homologous renin to the incubation mixture did not generate more angiotensin I from normal and bilaterally nephrectomized rat plasma which had been incubated before with an excess of rabbit renin. The isoelectric focusing profiles of plasma renin substrate from normal and bilaterally nephrectomized rats were almost identical using an excess of either rat or rabbit renin. It is concluded that there is no species-specific renin substrate for homologous renin in normal or bilaterally nephrectomized rats.

Existence of prokallikrein in the kidney. Its biochemical properties compared to three active glandular kallikreins from the kidney, serum, and urine of the rat.

Prokallikrein in the kidney was partially purified with immunoaffinity and DEAE Sephadex A-50 column chromatographies, and its biochemical properties were studied in comparison to three active glandular kallikreins purified from kidney, serum, and urine of the rat. The properties of the enzyme obtained by trypsin activation of prokallikrein were identical with those of active glandular kallikreins from the kidney, serum, and urine of the rat. Apparent molecular weights of prokallikrein, trypsin-activated kallikrein, active renal kallikrein, and glandular kallikrein in rat serum were 38,000 and of active urinary kallikrein, 37,000. Prokallikrein fraction was activated only by trypsin, but not by acidification, pepsin, and rat urinary esterase A treatments. Renal kallikrein, purified in the presence of soybean trypsin inhibitor (SBTI), contained 85% prokallikrein, but the enzymic fraction, purified in the absence of SBTI, contained 23% prokallikrein. Prokallikrein contents of urinary kallikrein and glandular kallikrein in rat serum were 16% and 20% respectively. These results suggest that prokallikrein is produced in the kidney and activated easily by a trypsin-like enzyme. Since rat serum contains active glandular kallikrein, kallikrein in the kidney may be secreted not only into the urine, but also into the blood.

Role of glandular kallikrein in the activation process of human plasma inactive renin.

Completely inactive renin was isolated from normal human plasma by DEAE-Sepharose column chromatography and Blue-Sepharose column chromatography. This inactive renin had a molecular weight of 54,000 daltons as determined by gel filtration on Ultrogel AcA 44. When the inactive renin was activated by trypsin, its molecular weight decreased to 48,000 daltons. The trypsin-activated renin differed from a native form of active renin in plasma with respect to molecular weight (active renin, 43,000), pI value (active renin, 5.20; trypsin-activated renin, 5.06), km value (active renin, 60 nmoles/liter; trypsin-activated renin, 89 nmoles/liter), Ki value for pepstatin A (active renin, 2.6 mumoles/liter; trypsin-activated renin 5.0 mumoles/liter) and pH profile for angiotensin formation. Glandular kallikrein (human urinary or pig pancreatic) did not activate the inactive renin. When the trypsin-activated renin was treated with glandular kallikrein, its activity was unchanged, but its molecular and kinetic properties except pI value (trypsin-activated kallikrein-treated renin, 4.82) coincided with those of a native form of active renin in plasma. These results indicate that glandular kallikrein does not directly activate inactive renin but participates in the activation process of inactive renin. The results also suggest that inactive renin in human plasma is a renin precursor.

Vascular smooth muscle calponin. A novel troponin T-like protein.

In a search for additional Ca2+ regulatory components in vascular smooth muscle, a novel troponin T-like protein was purified from bovine aorta smooth muscle. The isolated protein was separated into several isoforms on isoelectric focusing. The major isoelectric variants were focused in the pH region of 8.4 to 9.1. The protein had slightly different molecular masses in the Mr range of 35,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its molar ratio relative to tropomyosin in the muscle extract was estimated to be 0.9:1.0. The novel protein bound to the immobilized calmodulin and exhibited a number of common physicochemical properties with gizzard (Mr = 34,000) calmodulin-binding and F-actin-binding protein. The aorta and gizzard proteins were immunologically cross-reactive. Both proteins shared a common antigenic determinant with COOH-terminal segments of rabbit skeletal and bovine cardiac troponin T and bound to the immobilized smooth muscle tropomyosin. Both proteins interacted with rabbit skeletal troponin C in the presence and absence of Ca2+, but they did not interact with troponin I. These results suggest that the novel protein, which is designated calponin, may be a specialized component of smooth muscle thin filament involved in the regulation of contractile apparatus.

Statine-containing dipeptide and tripeptide inhibitors of human renin.

Dipeptide and tripeptide derivatives containing a statine residue were synthesized as inhibitors of human renin. ES-305, bis[(1-naphthyl)methyl]acetyl(BNMA)-histidyl-statine 2(S)-methylbutylamide was found to be a highly potent inhibitor of human renin with a Ki value of 1.7 X 10(-9) M. Dipeptide derivatives with the BNMA group at the N-terminal (BNMA-Val-Sta-isoleucinol [ES-313], BNMA-Leu-Sta-isoleucinol [ES-316], and BNMA-Nle-Sta-isoleucinol [ES-317]) had potencies against human renin that were similar to the potency of ES-305. All these dipeptide derivatives competitively inhibited human renin. The inhibitors were also potent against monkey renin but were less effective against renins from pig, goat, dog, rabbit, and rat. ES-305 had little effect on cathepsin D and pepsin at the concentration of 10(-5) M. The other derivatives showed detectable inhibition of cathepsin D (IC50, 10(-6) - 10(-7) M) and pepsin (10(-5) - 10(-6) M). All the compounds had little or no effect on trypsin, chymotrypsin, angiotensin converting enzyme, and urinary kallikrein at the concentration of 10(-5) M. Our results indicate that ES-305 is a highly potent and specific inhibitor of human renin. This compound is superior to other, previously described statine-containing renin inhibitors with respect to molecular size and enzyme specificity.

Blood pressure response to the Valsalva maneuver in pheochromocytoma and pseudopheochromocytoma.

To elucidate whether a difference in blood pressure reactivity exists between patients with pheochromocytoma (n = 8) and pseudopheochromocytoma (n = 22), we evaluated blood pressure changes during a Valsalva maneuver and baroreceptor reflex sensitivity. We also examined the effects of propranolol and prazosin on blood pressure reactivity during a Valsalva maneuver in patients with pseudopheochromocytoma. Pseudopheochromocytoma was defined as a paroxysmal rise in blood pressure accompanying pheochromocytoma-like symptoms and normal catecholamine values. The difference in systolic blood pressure between phase IV of the Valsalva maneuver and baseline (delta SBP) was markedly smaller in the pheochromocytoma patients (8.4 +/- 18.4 mm Hg) than in the essential hypertension patients (n = 30, 30.9 +/- 19.4 mm Hg) and normotensive control subjects (n = 10, 31.3 +/- 11.4 mm Hg), whereas delta SBP in the pseudopheochromocytoma patients (77.8 +/- 11.2 mm Hg) was markedly greater than in the other three groups. delta SBP was markedly suppressed by the administration of both propranolol and prazosin. Baroreceptor reflex sensitivity index was lower in the pheochromocytoma group than in the other three groups. In conclusion, blood pressure reactivity responses to a Valsalva maneuver are disparate between pheochromocytoma and pseudopheochromocytoma. The high blood pressure reactivity to a Valsalva maneuver in pseudopheochromocytoma is due to hyperactivity in both beta- and alpha 1-adrenergic receptor functions, and the low blood pressure reactivity to a Valsalva maneuver in pheochromocytoma seems to be mainly due to the desensitization of both adrenergic systems associated with chronic catecholamine excess. In addition, the impaired baroreceptor function in pheochromocytoma is partially responsible for it.

Highly potent and specific inhibitors of human renin.

Small peptide analogues representing the C-terminal portion of angiotensin I sequence were designed as inhibitors of human renin. Among synthesized compounds, benzyloxycarbonyl (-"Z")-(1-naphthyl)Ala-His-leucinal (ES-188), Z-(1-naphthyl)Ala-His-statine ethyl ester (ES-226), and Z-(1-naphthyl)Ala-His-statine 2-methylbutylamide (ES-254) markedly inhibited human and primate renins (inhibitory concentration, 50% [IC50], near 10(-7) M). These peptide analogues inhibited rabbit renin with one or two orders of magnitude less potency. They were very weak inhibitors of renins from pig, goat, dog, and rat. ES-188 had no discernible effect on cathepsin D, pepsin, or human angiotensin-converting enzyme at the concentration of 10(-4)M. ES-226 had little effect on the three enzymes at the concentration of 10(-5)M; however, ES-254 had a considerable inhibitory effect on cathepsin D (IC50 of 1.4 X 10(-5)M), pepsin (IC50 of 4.2 X 10(-5)M), and human angiotensin-converting enzyme (IC50 of 7.1 X 10(-6)M). Our results indicate that 1-naphthylalanine-containing tripeptide analogues are highly potent human renin inhibitors.

A highly potent and long-acting oral inhibitor of human renin.

An orally active renin inhibitor, ES 6864 (N-[(2R)-3-morpholinocarbonyl-2-(1-naphthylmethyl)propionyl]-(4- thiazolyl)-L-alanyl-cyclostatine-(2-morpholinoethyl)amide), was synthesized. ES 6864 was found to be a highly potent inhibitor of human renin with a Ki value of 7.3 x 10(-9) M. The compound competitively inhibited human renin. The inhibitor was also potent against monkey renin but was less effective against renins from pig, goat, dog, rabbit, and rat. ES 6864 did not inhibit cathepsin D, pepsin, trypsin, chymotrypsin, angiotensin converting enzyme, and urinary kallikrein at a concentration of 10(-5) M. ES 6864 was resistant to proteolytic actions of the enzymes in rat tissue homogenates (liver, kidney, pancreas, and small intestine). Oral administration of ES 6864 at 30 mg/kg to conscious, sodium-depleted marmosets produced a significant blood pressure reduction and almost complete inhibition of plasma renin activity, which persisted for 5 hours. Oral administration of ES 6864 also produced dose-related decreases of blood pressure in hog renin-infused rats, but the duration of action was much shorter than that in conscious marmosets. The parent compound in the blood following oral administration of ES 6864 to marmosets was confirmed directly by measuring the plasma concentration of ES 6864. These results enhance the possibility of developing renin inhibitors that can be used clinically.

ES-8891, an orally active inhibitor of human renin.

A newly synthesized orally active renin inhibitor, N-morpholinoacetyl-(1-naphthyl)-L-alanyl-(4-thiazolyl)-L-alanyl (3S,4S)-4-amino-3-hydroxy-5-cyclohexylpentanoyl-n-hexylamide (ES-8891), was found to be a highly potent competitive inhibitor of human renin with an inhibition constant of 1.1 nM. This inhibitor was also active against monkey renin, although there was less inhibition of renin in pig, rabbit, and rat. ES-8891 did not inhibit cathepsin D, pepsin, trypsin, chymotrypsin, angiotensin converting enzyme, and urinary kallikrein at a concentration of 10(-5) M. A single oral administration of ES-8891 (10 or 30 mg/kg) to conscious, sodium-depleted marmosets caused a dose-related decrease in plasma renin activity and blood pressure. ES-8891 (30 mg/kg) produced an 80% inhibition of plasma renin activity, which lasted for more than 6 hours. Kidney renin messenger RNA was not significantly changed 6 hours after oral administration of ES-8891 (30 mg/kg). A single oral administration of 240 mg ES-8891 to healthy human volunteers (n = 6) produced a significant inhibition of plasma renin activity (75% inhibition at 0.5 and 1 hour, 50% inhibition at 2 hours) with a good correlation of plasma levels of ES-8891. There were no significant changes in blood pressure or heart rate, and no adverse effects were observed. These results suggest that ES-8891 is an orally active human renin inhibitor that may be clinically useful.

Serum apolipoprotein A-I, A-II and B levels and their discriminative values in relatives of patients with coronary artery disease.

Serum cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C) and apolipoprotein (apo) A-I, A-II and B concentrations were measured in 109 first-degree relatives of patients with angiographically defined coronary artery disease (CAD). Age- and sex-matched healthy factory employees were chosen as a control group. Male relatives of the CAD patients had significantly higher serum triglyceride and apoB levels, and significantly lower serum HDL-C and apoA-I levels than the controls. Female relatives of the CAD patients also showed similar differences in serum HDL-C, apoA-I and apoB levels. Discriminant analysis indicated that apolipoproteins were better discriminators than lipids in both patients with CAD and their relatives. In univariate analysis, the best discriminator was apoB between male relatives and the controls, and apoA-I between female relatives and the controls. The percentage of exact classification achieved using three variables (serum cholesterol, triglyceride and HDL-C) was 74% in male relatives and 70% in female relatives. By adding variables of apoA-I and apoB, the percentage of correctly classified subjects was increased to 82% and 80%, respectively. These results indicate that serum apolipoprotein abnormalities are prevalent in relatives of the CAD patients. These abnormalities may explain the familial aggregation of CAD.

Clinical significance of measurements of serum apolipoprotein A-I, A-II and B in hypertriglyceridemic male patients with and without coronary artery disease.

To examine the relationship of hypertriglyceridemia to coronary artery disease (CAD), we measured serum cholesterol, triglyceride, high density lipoprotein cholesterol (HDL-C) and apolipoproteins (apo) A-I, A-II and B in 82 male patients with angiographically defined CAD and 140 age-matched healthy controls. The CAD patients had significantly lower apo A-I and A-II and HDL-C levels, but had higher apo B and triglyceride levels than the controls. After adjustments of apolipoproteins for serum triglyceride, CAD patients had significantly higher apo B and lower apo A-I and A-II levels than the controls. Discriminant analysis showed that apo B was the best discriminator and that apo A-I was next. In the normotriglyceridemic subgroup HDL-C also had a sufficient power for discrimination between CAD patients and the controls, but in the hypertriglyceridemic subgroup HDL-C had no discriminative power. Both apo A-I and B had significant discriminative power between CAD patients and the controls, independently of the serum triglyceride level. These results indicate that measurements of serum apo A-I and apo B are useful for the study of coronary risk factor in hypertriglyceridemic subjects. Finally, it is necessary to sub-classify dyslipoproteinemia by serum apolipoprotein levels for predicting the future occurrence of CAD in the general population.

Plasma lipids and lipoproteins in Japanese male patients with coronary artery disease and in their relatives.

Plasma cholesterol (CH), triglyceride (TG) and high density lipoprotein cholesterol (HDL-C) were measured in 92 consecutive Japanese male subjects undergoing diagnostic coronary cineangiography. Sixty-nine of them were classified as having coronary artery disease (CAD), the remaining 23 subjects were classified as having normal coronary arteries (NCA). The CAD group had significantly lower HDL-C and higher TG levels than the NCA group. However, there was no significant difference in plasma CH between the two groups. First-degree relatives of the CAD patients were also investigated. The male blood relatives of the CAD patients also had significantly lower HDL-C and higher TG levels than the non-blood male relatives and healthy control males. The female blood relatives, however, showed no significant differences from the non-blood female relatives and the healthy control females in plasma CH, TG and HDL-C levels. These results suggest that low HDL-C and hypertriglyceridemia are the prevalent coronary risk factors, rather than hypercholesterolemia, in a population with a low fat intake such as the Japanese, and that these lipid abnormalities are related to sex and genetic factors.

Kidney renin gene expression in spontaneously hypertensive rats.

We studied the expression of kidney renin gene in hypertensive animals by measuring the kidney renin messenger (m) RNA. The kidney renin mRNA was quantified by densitometric Northern blot analysis using a 32P-labelled rat renin genomic DNA fragment as a hybridization probe. Spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) were treated with a low-sodium diet plus furosemide, captopril or propranolol for a week. Plasma renin activity (PRA) in SHR and WKY was increased similarly by sodium depletion and by treatment with captopril. PRA in both strains was not decreased significantly by treatment with propranolol. Both sodium depletion and captopril treatment caused significant increases in the kidney renin mRNA in SHR and WKY. However, the increases in the kidney renin mRNA of SHR were greater than those in the corresponding WKY (SHR, 10.0- and 22.1-fold increases; WKY, 6.2- and 7.8-fold increases, respectively). Propranolol had no effect on the kidney renin gene expression in either WKY or SHR. These results indicate that SHR show an enhanced expression of the renin gene in the kidney compared with WKY in response to stimuli that increase renin release.