Homozygous whole body Cbs knockout in adult mice features minimal pathology during ageing despite severe homocysteinemia.

Deficiencies in Cystathionine-ß-synthase (CBS) lead to hyperhomocysteinemia (HHCy), which is considered a risk factor for cardiovascular, bone and neurological disease. Moreover, CBS is important for the production of cysteine, hydrogen sulfide (H2 S) and glutathione. Studying the biological role of CBS in adult mice has been severely hampered by embryological disturbances and perinatal mortality. To overcome these issues and assess the effects of whole-body CBS deficiency in adult mice, we engineered and characterized a Cre-inducible Cbs knockout model during ageing. No perinatal mortality occurred before Cbs-/- induction at 10 weeks of age. Mice were followed until 90 weeks of age and ablation of Cbs was confirmed in liver and kidney but not in brain. Severe HHCy was observed in Cbs-/- (289 ± 58 µM) but not in Cbs+/- or control mice (<10 µM). Cbs-/- showed impaired growth, facial alopecia, endothelial dysfunction in absence of increased mortality, and signs of liver or kidney damage. CBS expression in skin localized to sebaceous glands and epidermis, suggesting local effects of Cbs-/- on alopecia. Cbs-/- showed increased markers of oxidative stress and senescence but expression of other H2 S producing enzymes (CSE and 3-MST) was not affected. CBS deficiency severely impaired H2 S production capacity in liver, but not in brain or kidney. In summary, Cbs-/- mice presented a mild phenotype without mortality despite severe HHCy. The findings demonstrate that HHCy is not directly linked to development of end organ damage.

The Novel Compound Sul-121 Preserves Endothelial Function and Inhibits Progression of Kidney Damage in Type 2 Diabetes Mellitus in Mice.

Diabetic nephropathy is still a common complication of type 2 diabetes mellitus (T2DM) and improvement of endothelial dysfunction (ED) and inhibition of reactive oxygen species (ROS) are considered important targets for new therapies. Recently, we developed a new class of compounds (Sul compounds) which inhibit mitochondrial ROS production. Here, we tested the therapeutic effects of Sul-121 on ED and kidney damage in experimental T2DM. Diabetic db/db and lean mice were implanted with osmotic pumps delivering Sul-121 (2.2 mg/kg/day) or vehicle from age 10 to 18 weeks. Albuminuria, blood pressure, endothelial mediated relaxation, renal histology, plasma creatinine, and H2O2 levels were assessed. Sul-121 prevented progression of albuminuria and attenuated kidney damage in db/db, as evidenced by lower glomerular fibronectin expression (~50%), decreased focal glomerular sclerosis score (~40%) and normalization of glomerular size and kidney weight. Further, Sul-121 restored endothelium mediated vasorelaxation through increased production of Nitric Oxide production and normalized plasma H2O2 levels. Sul-121 treatment in lean mice demonstrated no observable major side-effects, indicating that Sul-121 is well tolerated. Our data show that Sul-121 inhibits progression of diabetic kidney damage via a mechanism that involves restoration of endothelial function and attenuation of oxidative stress.

Disruption of endothelial caveolae is associated with impairment of both NO- as well as EDHF in acetylcholine-induced relaxation depending on their relative contribution in different vascular beds.

Caveolae represent an important structural element involved in endothelial signal-transduction. The present study was designed to investigate the role of caveolae in endothelium-dependent relaxation of different vascular beds. Caveolae were disrupted by cholesterol depletion with filipin (4x10(-6) g L(-1)) or methyl-beta-cyclodextrin (MCD; 1x10(-3) mol L(-1)) and the effect on endothelium-dependent relaxation was studied in rat aorta, small renal arteries and mesenteric arteries in the absence and presence of L-NMMA. The contribution of NO and EDHF, respectively, to total relaxation in response to acetylcholine (ACh) gradually changed from aorta (71.2+/-6.1% and 28.8+/-6.1%), to renal arteries (48.6+/-6.4% and 51.4+/-6.4%) and to mesenteric arteries (9.1+/-4.0% and 90.9+/-4.1%). Electron microscopy confirmed filipin to decrease the number of endothelial caveolae in all vessels studied. Incubation with filipin inhibited endothelium-dependent relaxation induced by cumulative doses of ACh (3x10(-9)-10(-4) mol L(-1)) in all three vascular beds. In aorta, treatment with either filipin or MCD only inhibited the NO component, whereas in renal artery both NO and EDHF formation were affected. In contrast, in mesenteric arteries, filipin treatment only reduced EDHF formation. Disruption of endothelial caveolae is associated with the impairment of both NO and EDHF in acetylcholine-induced relaxation.

Acetylcholine stimulated dilatation and stretch induced myogenic constriction in mesenteric artery of rats with chronic heart failure.

Rats with chronic heart failure (CHF) develop increased myogenic constriction in mesenteric resistance arteries. Here we investigated increased myogenic constriction in relation to alterations in EDHF- and NO-mediated dilatation in CHF-rats. Male Spraque-Dawley rats were subjected to myocardial-infarction or sham-surgery. At 9-10 weeks after surgery, isolated mesenteric artery ring preparations were studied in a wire-myograph. Stretch-induced myogenic constriction was obtained by stepwise increase of the internal circumference diameter (0.5-1.2 L100). Cyclooxygenase- and eNOS-inhibitors were employed to study NO- and EDHF-mediated dilatation in response to acetylcholine. Rats with CHF (n=8), but not sham-rats (n=6), developed significant myogenic constriction. In addition, the contribution of endothelial dilator mediators was significantly altered in CHF-rats, with increased dependency on NO and decreased EDHF-mediated dilatation. Moreover, EDHF-mediated dilatation was inversely correlated with myogenic constriction in individual CHF-rats (r=-0.74, p=0.04). These data demonstrate increased myogenic constriction in mesenteric arteries of rats with CHF post-MI to be correlated to decreased EDHF-mediated dilatation. These findings extend the previous observation that myogenic constriction antagonizes EDHF-mediated dilatation in rat coronary artery under normal conditions, and suggests this relationship also to become functional in mesenteric arteries under pathophysiological conditions of CHF.

Male, female and management risk factors for non-return to service in Dutch mares.

The "effect" of stallion, mare and management-related factors on the odds of pregnancy per cycle in the horse were identified and quantified from the breeding records of Dutch Warmblood (n=4491), Friesian (n=1467) and Shetland-pony mares (n=3267) mated either naturally or by artificial insemination to one of the 88 stallions between 1992 and 1996. A mare was considered to be pregnant when she did not return to oestrous within 28 days of the last insemination. For Dutch Warmblood horses, the percentage of mares that did not return for service within 28 days (NR28) varied between studfarms and ranged from 61 to 82%. The NR28 for mares inseminated with fresh semen ranged from 67 to 74% and for mares inseminated with frozen/thawed semen this percentage was 59. Mares served at a second cycle had lower odds not to return than mares served at the third or subsequent cycle (OR=0.84). For Friesian horses, the NR28 for young mares was higher than that for older mares. Mares served before 1 May in any year had lower odds of non-return than mares served after 1 July (OR=0.69). The NR28 of mares inseminated once per cycle was 6% lower than that of mares inseminated three times or more per cycle. For Shetland ponies, the NR28 also varied between studfarms and ranged from 62 to 78%. Stallions < or =3 years old had lower odds of non-return compared to older stallion (> or =11) (OR=0.57). Mares served before 1 July had lower odds of non-return. Other significant factors for this breed were age of the mare, cycle number and insemination frequency. Stallion factors accounted for 5.9, 2.0 and 14.7% of the variation in the NR28 for Dutch Warmblood, Friesian horses and the Shetland ponies, respectively.

Exposure of progesterone receptors on the plasma membranes of stallion spermatozoa as a parameter for prediction of fertility.

Subfertility in stallions is attributed to the inability of spermatozoa to undergo the acrosome reaction in response to progesterone. In the present study, it was assessed whether there is a correlation between stallion fertility, defined on the basis of first cycle foaling rate and first cycle 'non-return rate', and the proportion of spermatozoa with exposed progesterone receptors on their plasma membranes. Semen from Dutch Warmblood (n=10) and Friesian (n=4) stallions was analysed. Progesterone 3-(o-carboxymethyl) oxime-BSA coupled with fluorescein isothiocyanate was used as a progesterone receptor probe and ethidium homodimer was used as a supravital stain. A high correlation was observed between the proportion of spermatozoa with exposed progesterone receptors and stallion fertility (r > 0.70; P < 0.01). This result indicates that exposure of progesterone receptors is a potential parameter for predicting stallion fertility.

[The prevention of pneumovagina and the effect of the Caslick operation on fertility: a retrospective study]. / Het voorkomen van pneumovagina en het effect van de Caslickoperatie op de fertiliteit: een retrospectief onderzoek.

Mares with a large vulvar orifice have an increased risk of pneumovagina, which is associated with reduced fertility. Treatment is by means of the Caslick operation, by which size of the vulvar orifice is reduced. Factors that influence the occurrence of pneumovagina are the age and fertility status of the mare. In this study the risk factors for pneumovagina and the effect of the Caslick operation on fertility were investigated. In 1994, 1995, and 1996 a total of 967 mares were sent for service at a stud. Of these mares, 116 underwent a Caslick operation. The operated mares were older than the non-operated mares. Mares younger than 5 years had a lower chance of pneumovagina than mares aged 5-12 years, but mares aged 13 and older had an increased chance. Foals and barren mares were more likely than maiden mares to develop pneumovagina. Fertility was 67% among the non-operated mares and 51% among the operated mares, even though, after the operation, the vulvar orifice of the operated mares was the same as that of the non-operated mares. This difference in fertility may be due to secondary endometritis that continues to exist after the operation. Further research is needed to determine whether mares with pneumovagina should also be treated for endometritis at the time of surgery.

Fertility of Shetland pony stallions used in different breeding systems: a retrospective study.

In horses reproductive performance is usually expressed as the foaling rate. This rate ranges from 40% to 80%. Three major factors contribute to this variation namely, the stallion, the mare and management. In this study, the performance of Shetland ponies kept in three different breeding systems was investigated retrospectively. In one breeding system, the stud farmer travelled with his stallion (n = 9) to the mare (system 1) while in another system, the stallion (n = 3) stayed at the stud farm and the mares came to the stallion (system 2). The last system was pasture breeding (system 3; n = 9). Each stallion participated in only one system. The average number of cycles per mare used for breeding did not differ significantly between systems 1 and 2. However, the number of matings per cycle was higher in system 2 than in system 1. The average number of mares serviced per stallion was 91, 50, and 17 for systems 1, 2 and 3, respectively. Mares mated in pasture had a 2.8-fold higher chance (p < 0.05) of having a foal the next season than the mares mated under systems 1 and 2. The foaling rate per season was 58%, 48%, and 80% for systems 1, 2 and 3, respectively. Management aspects play an important role in the relatively low foaling percentages of systems 1 and 2.

Dyslipidemia and endothelium-dependent relaxation in internal mammary arteries used for coronary bypass surgery.

OBJECTIVE: Impairment of endothelium-dependent relaxation is related to dyslipidemia and may be an early marker for atherosclerosis in angiographically smooth arteries. The aim of the present study was to relate preoperative serum lipids to endothelium-dependent relaxation in internal mammary arteries of patients undergoing coronary bypass surgery. METHODS: The study group consisted of 37 patients, from whom segments of the internal mammary artery were obtained during surgery. Measurements of endothelium-dependent relaxation were performed in organ baths by adding methacholine (10 nM-10 microM). RESULTS: All internal mammary arteries dilated in response to methacholine, ranging from 4 to 112% of the precontraction to 10 mumol phenylephrine. In a multiple regression model, increased total serum cholesterol appeared to be the best predictor for impaired endothelium-dependent relaxation. A 1 mmol increase of total cholesterol was associated with a 11.2% decrease of endothelium-dependent relaxation (P = 0.006). When total cholesterol was omitted from the model, LDL-cholesterol became the best predictor of endothelium-dependent relaxation (regression coefficient 10.3%/mmol; P = 0.02). No other variable was significantly associated with endothelium-dependent relaxation, and none of the preoperative variables was associated with endothelium-independent relaxation, expressed as the response to sodium nitrite (10 mM). CONCLUSION: Our study showed that endothelium-dependent relaxation in apparently non-diseased internal mammary arteries used for coronary bypass surgery was independently related to preoperative (LDL)-cholesterol levels.

Strain of the musculus interosseus medius and its rami extensorii in the horse, deduced from in vivo kinematics.

The in vivo strains of the musculus interosseus medius (suspensory ligament) and its rami extensorii (extensor branches) in the forelimb of the horse were determined from angular changes of the metacarpophalangeal and the distal interphalangeal joints. For this purpose, regression models were fitted to strains and joint angle combinations measured in in vitro limb loading experiments. The in vivo strains were computed from the kinematics of 8 horses at the walk, the trot and the canter. It was found that the extensor branches were strained about 1.0% at hoof impact, which indicates that they passively extend the interphalangeal joints just prior to impact and prevent flexion of the pastern joint just thereafter. The maximal strain of the suspensory ligament amounted to 3.4% at the walk, 5.6% at the trot and 6.3% at a slow canter.