Amlodipine accelerates bone healing in a stable closed femoral fracture model in mice.

Calcium channel blockers (CCBs), which are widely used in the treatment of hypertension, have been shown to influence bone metabolism. However, there is little information on whether CCBs also influence the process of fracture healing. Therefore, the effect of the CCB amlodipine on bone healing was studied in a stable closed fracture model in mice using intramedullary screw fixation. Bone healing was investigated by radiology, biomechanics, histomorphometry and Western blot analysis 2 and 5 weeks after fracture healing. Animals were treated daily (post operatively) per os using a gavage with amlodipine low dose (1 mg/ kg body weight, n = 20), amlodipine high dose (3 mg/kg body weight, n = 20) or vehicle (NaCl) (control, n = 20) serving as a negative control. At 2 and 5 weeks, histomorphometric analysis revealed a significantly larger amount of bone tissue within the callus of amlodipine low-dose- and high-dose-treated animals when compared to controls. This was associated with a smaller amount of cartilaginous and fibrous tissue, indicating an acceleration of fracture healing. Biomechanics showed a slightly, but not significantly, higher bending stiffness in amlodipine low-dose- and high-dose-treated animals. Western blot analysis revealed a significantly increased expression of bone morphogenetic protein (BMP)-2 and vascular endothelial growth factor (VEGF). Moreover, the analysis showed a 5-fold higher expression of osteoprotegerin (OPG) and a 10-fold elevated expression of the receptor activator of NF-κB ligand (RANKL), indicating an increased bone turnover. These findings demonstrated that amlodipine accelerated fracture healing by stimulating bone formation, callus remodelling and osteoclast activity.

Parathyroid hormone [1-34] improves articular cartilage surface architecture and integration and subchondral bone reconstitution in osteochondral defects in vivo.

OBJECTIVE: The 1-34 amino acid segment of the parathyroid hormone (PTH [1-34]) mediates anabolic effects in chondrocytes and osteocytes. The aim of this study was to investigate whether systemic application of PTH [1-34] improves the repair of non-osteoarthritic, focal osteochondral defects in vivo. DESIGN: Standardized cylindrical osteochondral defects were bilaterally created in the femoral trochlea of rabbits (n = 8). Daily subcutaneous injections of 10 µg PTH [1-34]/kg were given to the treatment group (n = 4) for 6 weeks, controls (n = 4) received saline. Articular cartilage repair was evaluated by macroscopic, biochemical, histological and immunohistochemical analyses. Reconstitution of the subchondral bone was assessed by micro-computed tomography. Effects of PTH [1-34] on synovial membrane, apoptosis, and expression of the PTH receptor (PTH1R) were determined. RESULTS: Systemic PTH [1-34] increased PTH1R expression on both, chondrocytes and osteocytes within the repair tissue. PTH [1-34] ameliorated the macro- and microscopic aspect of the cartilaginous repair tissue. It also enhanced the thickness of the subchondral bone plate and the microarchitecture of the subarticular spongiosa within the defects. No significant correlations were established between these coexistent processes. Apoptotic levels, synovial membrane, biochemical composition of the repair tissue, and type-I/II collagen immunoreactivity remained unaffected. CONCLUSIONS: PTH [1-34] emerges as a promising agent in the treatment of focal osteochondral defects as its systemic administration simultaneously stimulates articular cartilage and subchondral bone repair. Importantly, both time-dependent mechanisms of repair did not correlate significantly at this early time point and need to be followed over prolonged observation periods.

Anti-angiogenic treatment strategies for the therapy of endometriosis.

BACKGROUND Angiogenesis, i.e. the development of new blood vessels from pre-existing ones, represents an integral part in the pathogenesis of endometriosis. During the last decade, an increasing number of studies have therefore focused on the anti-angiogenic treatment of the disease. The present review provides a systematic overview of these studies and critically discusses the future role of anti-angiogenic therapy in the multimodal management of endometriosis. METHODS Literature searches were performed in PubMed, MEDLINE and ISI Web of Knowledge for original articles published before the end of March 2012, written in the English language and focusing on anti-angiogenic approaches for the therapy of endometriosis. The searches included both animal and human studies. RESULTS Numerous compounds of different substance groups have been shown to exert anti-angiogenic effects on endometriotic lesions under experimental in vitro and in vivo conditions. These include growth factor inhibitors, endogenous angiogenesis inhibitors, fumagillin analogues, statins, cyclo-oxygenase-2 inhibitors, phytochemical compounds, immunomodulators, dopamine agonists, peroxisome proliferator-activated receptor agonists, progestins, danazol and gonadotropin-releasing hormone (GnRH) agonists. However, clinical evidence for their efficacy in anti-angiogenic endometriosis therapy is still lacking. CONCLUSIONS Anti-angiogenic compounds hold great promise for the future treatment of endometriosis because they may inhibit the establishment of new endometriotic lesions in early stages of the disease or after surgical treatment. Further experimental studies, controlled clinical trials in particular, are required now to clarify which compounds fulfil these expectations without inducing severe side effects in patients with endometriosis.

Xanthohumol inhibits growth and vascularization of developing endometriotic lesions.

BACKGROUND: Xanthohumol is a prenylated flavonoid isolated from hops, which is known to act as a pleiotropic cancer chemopreventive agent owing to its anti-proliferative, anti-inflammatory and anti-angiogenic properties. In the present study, we analyzed, for the first time, whether this dietary compound may also be used for the treatment of endometriosis. METHODS: Peritoneal and mesenteric endometriotic lesions were surgically induced in BALB/c mice by uterine tissue transplantation into the abdominal cavity. The animals were treated daily with 100 µM xanthohumol (n= 8) or vehicle (control, n= 8) via the drinking water, starting 3 days before tissue transplantations. Lesion growth, cyst formation and vascularization were subsequently analyzed by means of high-resolution ultrasound imaging (at Day 0 and then once per week for 28 days), caliper measurements, western blotting, histology and immunohistochemistry over 4 weeks. RESULTS: In the treatment and control groups, uterine grafts developed typical endometriotic lesions with cyst-like dilated glands surrounded by a vascularized endometrial stroma. However, xanthohumol efficiently decreased the size of these lesions at Day 28, independent of their localization within the peritoneal cavity, compared with control (peritoneal: P =0.041; mesenteric: P =0.038). This was associated with a reduced level of phosphoinositide 3-kinase protein. Moreover, vascularization of xanthohumol-treated lesions was suppressed, as indicated by a significantly lower microvessel density at Day 28 when compared with vehicle-treated controls (peritoneal: P =0.026; mesenteric: P =0.004). Additional analyses revealed that treatment with xanthohumol did not affect the histomorphology, proliferation and vascularization of the uterine horns and ovaries. CONCLUSIONS: Taken together, these experimental findings suggest that xanthohumol inhibits the development of endometriotic lesions in mice without inducing serious side effects in the reproductive organs. Thus, xanthohumol represents a promising dietary phytochemical that, after further testing, may be considered for the use in the selective treatment of endometriotic lesions.

Hyperhomocysteinemia is associated with impaired fracture healing in mice.

Hyperhomocysteinemia (HHCY) has been shown to disturb bone metabolism and to increase the incidence of osteoporosis and osteoporotic fractures. However, there is a complete lack of information on whether these metabolic alterations affect bone repair. The aim of this study was to analyze the impact of HHCY on fracture healing. One group of mice was fed a homocystine-supplemented diet (n = 12), whereas another group received the accordant standard diet for control (n = 13). Four weeks after the stable fixation of a closed femoral fracture, animals were killed to prepare bones for histomorphometric and biomechanical analyses. In addition, blood samples were obtained to evaluate serum concentration of homocysteine (HCY). Quantitative analysis of blood samples revealed severe HHCY as indicated by significantly increased serum concentrations of HCY in animals fed the homocystine-supplemented diet (102.2 +/- 64.5 micromol/l) compared to controls (2.8 +/- 1.5 micromol/l). Biomechanical evaluation of bone repair revealed significantly decreased bending stiffness of the femora of homocystine-fed animals (45.5 +/- 18.2 N/mm) compared with controls (64.6 +/- 15.8 N/mm). Histomorphometric analysis demonstrated a slightly smaller callus diameter in HHCY animals but no significant differences in the tissue composition of the callus. In conclusion, the homocystine-supplemented diet leads to severe HHCY, which is associated with an impaired biomechanical quality of the healing bone.

An old dream revitalised: preconditioning strategies to protect surgical flaps from critical ischaemia and ischaemia-reperfusion injury.

The prevention of ischaemia and the adequate restitution of blood flow to ischaemic tissue are pivotal to halt the progression of cellular injury associated with decreased oxygen and nutrient supply. Accordingly, the search for novel strategies which aim at preventing ischaemia-reperfusion-induced tissue damage is still of major interest in flap surgery. Preconditioning represents an elegant approach to render the tissue more resistant against deleterious ischaemic insults. For many decades, 'surgical delay' has been the standard method of tissue preconditioning. During the last 10 years, ischaemic preconditioning was added to the repertoire of plastic surgeons to protect flaps from ischaemic necrosis. The invasiveness and expenditure of time of these procedures, however, have always been major drawbacks, hindering a wide distribution in clinical practice. Consequently, the motivation has all along been to further refine and simplify protective strategies. Recent experimental studies have now shown that efficient protection from ischaemic necrosis can also be achieved by remote preconditioning or pretreatment with chemical agents and growth factors, which mimic the action of surgical delay and ischaemic preconditioning. In addition, the local application of unspecific stressors, including both heating and cooling, have been shown to effectively improve flap microcirculation and, thus, tissue survival. In view of successful translational research, it is now time that the efficacy of these novel preconditioning procedures is proven in prospective randomised clinical trials.

Revascularization and microcirculation of freely grafted islets of Langerhans.

A considerable number of experimental studies have demonstrated that the reestablishment of an appropriate microvascular supply is an essential prerequisite for successful pancreatic islet transplantation. Freely transplanted islets show the first signs of angiogenesis (i.e., capillary sprout formation and protrusion) as early as 2 days after transplantation, and the entire vascularization process is completed after 10 to 14 days. Cryopreservation and culture of the isolated islets before transplantation and hyperglycemia of the transplant recipient seem not to affect the vascularization process essentially. In addition, immunosuppressive drugs, such as cyclosporin A and 15-deoxyspergualin, do not or only slightly inhibit revascularization of syngeneic islets; however, they are not able to prevent completely xenograft-induced microvascular perfusion failure. In contrast, novel immunosuppressants (e.g., RS-61443) or dietary supplementation of the antioxidant vitamin E were shown to prevent microvascular graft rejection almost completely, including leukocyte recruitment and capillary perfusion failure. Thus the development of novel strategies to improve posttransplant islet function should include concepts that accelerate the vascularization process and protect the newly formed microvasculature from rejection-mediated injury. The improvement of islet graft vascularization and the maintenance of adequate microvascular perfusion will contribute to the increased success of pancreatic islet transplantation.

Antithrombin reduces leukocyte adhesion during chronic endotoxemia by modulation of the cyclooxygenase pathway.

Antithrombin (AT) is known as the most important natural inhibitor of thrombin activity and has been shown to improve distinct clinical parameters during the course of septic (endotoxin)-induced multiple organ dysfunction. We hypothesized that AT acts by inhibiting leukocyte activation and microvascular injury via the promotion of endothelial release of PGI(2), and therefore, we studied the effects of AT on leukocyte/endothelial cell interaction and microvascular perfusion during endotoxemia. In a skinfold preparation of Syrian hamsters, severe endotoxemia was induced by repeated administration of endotoxin intravenously [lipopolysaccharide (LPS), Escherichia coli, 2 mg/kg] at 0 and 48 h. AT (250 IU/kg) was administered intravenously at 0, 24, and 48 h (n = 6, AT group). In control animals (n = 5, control), LPS was given without AT supplementation. By intravital fluorescence microscopy, leukocyte-endothelial cell interaction and functional capillary density (FCD; measure of capillary perfusion) were analyzed during a 72-h period after the first LPS injection. AT significantly attenuated LPS-induced arteriolar and venular leukocyte adherence after both the first and the second LPS injection [P < 0.01, measures analysis of variance (MANOVA)]. In parallel, AT was effective in preventing LPS-induced depression of FCD after the first and the second LPS administration (P < 0.05, MANOVA). By pretreatment with the cyclooxygenase inhibitor indomethacin (n = 6), effects of AT on leukocyte adherence and FCD were found completely abolished. Thus our study indicates that AT exerts its beneficial effects in endotoxemia by reducing leukocyte-endothelial cell interaction and microvascular perfusion failure probably via liberation of prostacyclin from endothelial cells.

Attenuation of microvascular reperfusion injury in rat pancreas transplantation by L-arginine.

BACKGROUND: Despite improving results, exocrine complications remain a major challenge in clinical pancreas transplantation. The etiology of posttransplantation pancreatitis relates almost certainly to cold ischemia/reperfusion-induced microvascular injury with an imbalance of vasoconstricting and vasodilating mediators due to endothelial dysfunction. We therefore studied the effectiveness of a nitric oxide donor on postischemic microvascular reperfusion injury after pancreas transplantation. METHODS: Heterotopic isogeneic pancreaticoduodenal transplantation was performed in spontaneously breathing, chloralhydrate-anesthetized Sprague Dawley rats after 16 hr (n=5) of cold storage of the graft in 4 degrees C histidine-tryptophane-ketoglutarate solution. An additional five animals received L-arginine immediately before (50 mg/kg i.v.) and during the first 30 min of reperfusion (100 mg/kg i.v.). Five animals that did not undergo transplantation served as controls. Intravital fluorescence microscopy was used for analysis of functional capillary density, capillary diameters, and capillary red blood cell velocity in exocrine pancreatic tissue during 120 min of reperfusion. Histology served for quantitative assessment of inflammatory response (leukocytic tissue infiltration) and endothelial disintegration (edema formation). RESULTS: In L-arginine-treated animals, functional capillary density of exocrine tissue of pancreatic grafts was found slightly higher after 30 and 60 min, and significantly higher after 120 min of postischemic reperfusion compared with untreated pancreatic grafts. This was accompanied by a significant increase of capillary diameters. In parallel, pancreatic histology revealed significant attenuation of both leukocytic tissue infiltration and edema formation in the L-arginine-treated animals when compared with the nontreated controls. CONCLUSIONS: Besides reduction of leukocyte-dependent microvascular injury, L-arginine improves postischemic microvascular reperfusion, supposedly by capillary dilatation. Thus, our results suggest that supplement of nitric oxide during reperfusion is effective in attenuating exocrine microvascular reperfusion injury.

Do vitamin E supplements in diets for laboratory animals jeopardize findings in animal models of disease?

Vitamin E has been supplemented to the diets of farm animals to improve fertility, health, growth rates and quality of animal products. Because of the positive experience obtained in farm animals, vitamin E has been added in increasing amounts to the diets of laboratory animals. Today, vitamin E levels in standard rodent maintenance diets range from 30 mg/kg (France, United States), 90-120 mg/kg (Netherlands, United Kingdom) to as much as 200 mg/kg (Germany). While increasing fertility and health of laboratory animals, these vitamin E supplements affect diverse pathophysiological conditions and thus the outcome of animal models of disease. Because of the large variability of vitamin E levels between laboratories within and between different countries, results obtained in established animal models may no longer be comparable and/or reproducible. Researchers should be aware of these vitamin E supplements and carefully control for potential effects in their respective animal models that involve--or may involve--the generation of reactive oxygen species.

[Reduction of inflammatory response after flap transfer by pretreatment with local heat shock]. / Reduktion der inflammatorischen Antwort nach Lappentransfer durch lokale Hitzeschock-Vorbehandlung.

In a rat model of free flap transfer local heat shock-priming is able to prevent ischemia/reperfusion-induced inflammatory response by expression of heat shock protein-32.

Prevention of pancreatic islet xenograft rejection by dietary vitamin E.

In pancreatic islet transplantation, the adhesion of activated leukocytes to endothelial cells and the loss of microvascular integrity represent the critical microcirculatory events, which promote loss of graft function due to rejection. With the view that oxygen radicals may contribute to graft rejection, we studied the effect of the antioxidant vitamin E on microvascular rejection of islet grafts. Islets were transplanted syngeneically and xenogeneically (rat) into dorsal skin-fold chambers of hamsters, which received a non-vitamin-E-supplemented laboratory chow. Treated animals with xenografts were fed with a diet supplemented with vitamin E in a low (150 mg/kg) and high (8000 mg/kg) concentration. Intravital fluorescence microscopy demonstrated complete vascularization of syngeneic grafts at day 10 after transplantation, intact islet microcirculation at day 20 with a functional capillary density of 653 +/- 6 cm-1, and only few leukocytes adherent to the endothelial lining of the islets' microvasculature (88 +/- 23 mm-2). Xenogeneic islets showed initial signs of rejection at day 6, including adhesion of leukocytes to the microvascular endothelium (610 +/- 110 mm-2) and loss of endothelial integrity. After 20 days, functional capillary density was significantly lower (173 +/- 68 cm-1) when compared with syngeneic grafts, indicating failure of graft acceptance. Supplementation of the diet with low and high concentrations of vitamin E resulted in a significant (P < 0.05) reduction of xenograft leukocyte-endothelium interaction (146 +/- 29 mm-2 and 109 +/- 42 mm-2) at day 6 after transplantation and and adequate development of functional capillary density at day 20 (478 +/- 36 cm-1 and 539 +/- 86 cm-1; P < 0.05), indicating prevention of microvascular rejection. We conclude that dietary supplementation of the lipophilic antioxidant vitamin E attenuates leukocyte-endothelial cell interactions, preserves microvascular integrity, and thus inhibits microvascular rejection in a dose-dependent fashion. Our study underscores the pivotal mediator role of reactive oxygen species in islet xenograft rejection and, furthermore, suggests that dietary vitamin E may act as an adjunct anti-rejection treatment in clinical islet transplantation.