Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth.

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/ß-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Osteocytic connexin 43 is not required for the increase in bone mass induced by intermittent PTH administration in male mice.

OBJECTIVE: To investigate whether osteocytic connexin 43 (Cx43) is required for the bone response to intermittent PTH administration, and whether the connexin is involved in maintaining the bone matrix. METHODS: Human PTH(1-34) was injected to adult male mice expressing (Cx43(fl/fl)) or not osteocytic Cx43 (Cx43(fl/fl);DMP1-8kb-Cre) daily (100 µg/kg/d) for 14 days. RESULTS: Cx43(fl/fl);DMP1-8kb-Cre mice have no difference in body weight and BMD from 1 to 4 months of age. Intermittent PTH administration increased BMD and BV/TV and induced a similar increase in type I collagen, alkaline phosphatase, runx2, osteocalcin, and bone sialoprotein expression in mice from both genotypes. On the other hand, osteocytic deletion of Cx43 did not alter mRNA levels of glycosaminoglycans, proteoglycans, collagens and osteoblast-related genes. In addition, expression of collagens assessed by immunohistochemistry was not affected by deleting osteocytic Cx43. However, PTH administration increased type II collagen only in Cx43(fl/fl) control mice, whereas hormone increased type I collagen expression only in Cx43(fl/fl);DMP1-8kb-Cre mice. Furthermore, PTH increased maturity of collagen fibers in control, but not in Cx43-deficient mice. CONCLUSION: Expression of Cx43 in osteocytes is dispensable for bone anabolism induced by intermittent PTH administration; but it can modulate, at least in part, the effect of PTH on the bone matrix environment.

Osteoblastic protein tyrosine phosphatases inhibition and connexin 43 phosphorylation by alendronate.

Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [(3)H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na3VO4, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.

[Clinical signs of endothelial disorders in patients with severe sepsis].

A clinical-experimental study was carried out. The objective was to find some regularities in endothelial disorder progression in patients with severe sepsis and to evaluate clinical efficacy of some methods of hepatic protection. Experimental part of work was carried out on 59 mice with induced peritonitis. Obtained data shows early emergence of lung disorders that precede changes in hepatic tissue. Clinical part of work included 181 patient with severe sepsis. It was noted that acute respiratory distress syndrome symptoms occurred earlier than hepatic dysfunction, if the latter joints, it aggravates the patients status and worsens the prognosis. Use of Heptral (Ademetionine) and Ketamine in order to protect liver is a clinically effective method which makes possible to decrease the lethality.

Role of connexin 43 in the mechanism of action of alendronate: dissociation of anti-apoptotic and proliferative signaling pathways.

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.

A bisphosphonate that does not affect osteoclasts prevents osteoblast and osteocyte apoptosis and the loss of bone strength induced by glucocorticoids in mice.

Although a major effect of bisphosphonates on bone is inhibition of resorption resulting from their ability to interfere with osteoclast function, these agents also prevent osteoblast and osteocyte apoptosis in vitro and in vivo. However, the contribution of the latter property to the overall beneficial effects of the drugs on bone remains unknown. We compared herein the action on glucocorticoid-induced bone disease of the classical bisphosphonate alendronate with that of IG9402, a bisphosphonate analog that preserves osteoblast and osteocyte viability but does not induce osteoclast apoptosis in vitro. The bisphosphonates were injected daily (2.3 µmol/kg) to 5-month-old Swiss Webster mice (6-11 per group), starting 3 days before implantation of pellets releasing the glucocorticoid prednisolone (2.1 mg/kg/day). IG9402 did not affect levels of circulating C-telopeptide or osteocalcin, markers of resorption and formation, respectively, nor did it decrease mRNA levels of osteocalcin or collagen 1a1 in bone. On the other hand, alendronate decreased all these parameters. Moreover, IG9402 did not reduce cancellous mineralizing surface, mineral apposition rate, or bone formation rate, whereas alendronate induced a decrease in each of these bone formation measures. These findings demonstrate that, in contrast to alendronate, IG9402 does not inhibit bone turnover. Both alendronate and IG9402, on the other hand, activated survival kinase signaling in vivo, as evidenced by induction of ERK phosphorylation in bone. Furthermore, both bisphosphonates prevented the increase in osteoblast and osteocyte apoptosis as well as the decrease in vertebral bone mass and strength induced by glucocorticoids. We conclude that a bisphosphonate that does not affect osteoclasts prevents osteoblast and osteocyte apoptosis and the loss of bone strength induced by glucocorticoids in mice.

[The prognostic role of interleukin-6 in peritoneal exudate of patients with diffuse peritonitis].

A prospective longitudinal controlled investigation of 189 patients with secondary diffuse peritonitis was performed. A correlation between the levelof interleuikin-6 of peritoneal exudate, the severity of the patients' state, the number of complications and outcome of the course of the disease was established. The maximal number of complications and unfavorable outcomes falls on the peak value ofinterleuikin-6.

[Relaparotomy in patients with diffuse purulent peritonitis, aspects of aggressiveness].

The lengthwise prospective investigation included 189 patients with diffuse purulent peritonitis for studying the influence of operative trauma on the indices of blood circulation, endocrine system, liver function, intensity of systemic inflammation syndrome as well as effects of high intraperitoneal pressure on the circulation system and on the outcome of the disease as a whole. The investigation has revealed a correlation between the survival rate and the number of reoperations. It was shown that the greater the number of reoperations the higher the risk of the development of the systemic inflammation syndrome, organic dysfunctions and number of complications.

[Diagnosis of hepatic failure at the patients with abdominal sepsis].

Results of prospective study of 398 patients with abdominal sepsis are analyzed. It is demonstrated that acute hepatic failure occurs at 45% of these patients and it is direct cause of death at 28% of them. Blood level of triglycerides and alkaline phosphatase are reliable markers of hepatic dysfunction at abdominal sepsis. Concentrations of these markers are associated with severity of patients and systemic inflammation syndrome, and also with the number of performed laparotomies.

[Clinical significance of inflammation markers in abdominal sepsis patients].

Possibilities of using C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), lactoferrin (LF) and sorption ability of erythrocytes (SAE) as markers of the severity, prognosis and criteria of effectiveness of treatment were studied in 334 patients with abdominal sepsis. The investigation of the sepsis marker dynamics has shown that fast kinetics of CRP and PCT makes it impossible to assess the prognosis and effectiveness of treatment using these markers. IL-6, LF and SAE are the only ones to be used for this purpose.

[Dysfunction of the endocrine system in patients with abdominal sepsis].

The article presents results of a prospective investigation of 270 patients with abdominal sepsis. Three variants of endocrine impairments were established which correlated with the intensity of syndrome of inflammatory response and severity of the patients' state. In addition, possible transition of one variant of the endocrine dysfunction into another was proved in case of increased number of programmed sanitation relaparotomies.

[Epidemiology of abdominal sepsis].

An analysis of 2376 case histories of patients with surgical infections has shown that in 827 (34.8%) patients the course of main disease was complicated by sepsis. The most frequent causes of the development of sepsis were surgical infections with localization in the abdominal cavity, lungs and mediastinum. Abdominal sepsis was diagnosed in 398 (41.7%) of patients with peritonitis. The annual growth of this category of patients was 8.3%. The outcome of abdominal sepsis was shown to depend on the score number according to scales APACHE II and SOFA, and on the timely operative treatment and beginning of antibacterial therapy.

[Course and outcome of surgical infections complicated with sepsis].

A retrospective study of 594 medical histories of patients with surgical infections suggested diagnosis of sepsis in 18.2% cases according to ACCP/SCCM criteria (1992). Pulmonary and abdominal sepsis prevailed and led to higher lethality. Sepsis developed most often as nosocomial infection caused primarily by Acinetobacter spp. and Ps.aeruginosa. Most patients were males aged from 40 to 60 years. The main causes of lethality were septic shock (49.3%) and multiple organ failure syndrome (21%). Time of surgical sanation, antibacterial therapy and its adequacy correlated with the disease outcome.

Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hormonal control of osteoblastogenesis.

Both chronic excess of PTH, as in hyperparathyroidism, and intermittent elevation of PTH (by daily injections) increase the number of osteoblasts; albeit, the former is associated with bone catabolism and the later with bone anabolism. Intermittent PTH increases osteoblast number by attenuating osteoblast apoptosis, an effect that requires the transcription factor Runx2. However, chronic elevation of PTH does not affect osteoblast apoptosis because it stimulates the proteasomal degradation of Runx2. Here, we studied the effects of PTH on Sost, a Runx2 target gene expressed in osteocytes (former osteoblasts embedded in the bone matrix), which antagonizes the pro-osteoblastogenic actions of bone morphogenetic proteins and Wnts. We report that continuous infusion of PTH to mice for 4 d decreased Sost mRNA expression in vertebral bone by 80-90%. This effect was accompanied by a comparable reduction of sclerostin, the product of Sost, in osteocytes, as determined by quantitative immunoblot analysis of bone extracts and by immunostaining. In contrast, a single injection of PTH caused a transient 50% reduction in Sost mRNA at 2 h, but four daily injections had no effect on Sost mRNA or sclerostin. PTH strongly decreased Sost expression in osteocytes formed in primary cultures of neonatal murine calvaria cells as well as in osteocytic MLO-A5 cells, demonstrating a direct effect of PTH on this cell type. These results, together with evidence that sclerostin antagonizes bone morphogenetic proteins and Wnts, strongly suggest that suppression of Sost by PTH represents a novel mechanism for hormonal control of osteoblastogenesis mediated by osteocytes.

Mechanical stimulation prevents osteocyte apoptosis: requirement of integrins, Src kinases, and ERKs.

Osteocytes, former osteoblasts entombed in the bone matrix, form an extensive cell communication network that is thought to detect microdamage and mechanical strains and to transmit signals leading to repair and compensatory bone augmentation or reduction. Bone active hormones and drugs control the integrity of this network by regulating osteocyte apoptosis, which might be a determinant of bone strength. Herein we demonstrate that mechanical stimulation by stretching activates the ERKs, which in turn are responsible for the attenuation of osteocyte apoptosis. The effect of osteocyte stretching is transmitted by integrins and cytoskeletal and catalytic molecules, such as Src kinases. Stretch-induced antiapoptosis also requires nuclear translocation of ERKs and new gene transcription. The evidence linking mechanical stimulation, activation of an integrin/cytoskeleton/Src/ERK signaling pathway, and osteocyte survival provides a mechanistic basis for the profound role of mechanical forces, or lack thereof, on skeletal health and disease.

Reversal of bone loss in mice by nongenotropic signaling of sex steroids.

We show that sex steroids protect the adult murine skeleton through a mechanism that is distinct from that used to preserve the mass and function of reproductive organs. The classical genotropic actions of sex steroid receptors are dispensable for their bone protective effects, but essential for their effects on reproductive tissues. A synthetic ligand (4-estren-3alpha,17beta-diol) that reproduces the nongenotropic effects of sex steroids, without affecting classical transcription, increases bone mass and strength in ovariectomized females above the level of the estrogen-replete state and is at least as effective as dihydrotestosterone in orchidectomized males, without affecting reproductive organs. Such ligands merit investigation as potential therapeutic alternatives to hormone replacement for osteoporosis in both women and men [corrected].

Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro.

Bisphosphonates (BPs) are analogues of pyrophosphate, which are widely used for the treatment of different pathologies associated with imbalances in bone turnover. Recent evidence suggested that cells of the osteoblastic lineage might be targets of the action of BPs. The objective of this work was to determine whether BPs induce proliferation of osteoblasts and whether this action involves activation of the extracellular signal-regulated kinases (ERKs). We have shown that three different BPs (olpadronate, pamidronate, and etidronate) induce proliferation in calvaria-derived osteoblasts and ROS 17/2.8 as measured by cell count and by [3H]thymidine uptake. Osteoblast proliferation induced by all BPs diminished to control levels in the presence of U0126, a specific inhibitor of the upstream kinase MEK 1 responsible for ERK phosphorylation. Consistent with this, BPs induced ERK activation as assessed by in-gel kinase assays. Phosphorylation of ERK1/2 was induced by the BPs olpadronate and pamidronate within 30 s, followed by rapid dephosphorylation, whereas etidronate induced phosphorylation of ERKs only after 90 s of incubation and returned to basal levels within 15-30 minutes. In addition, both BP-induced cell proliferation and ERK phosphorylation were reduced to basal levels in the presence of nifedipine, an L-type voltage-sensitive calcium channel (VSCC) inhibitor. These results show that BP-induced proliferation of osteoblastic cells is mediated by activation of ERKs and suggest that this effect requires influx of Ca2+ from the extracellular space through calcium channels.

Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity.

The relationship of the classical receptors and their transcriptional activity to nongenotropic effects of steroid hormones is unknown. We demonstrate herein a novel paradigm of sex steroid action on osteoblasts, osteocytes, embryonic fibroblasts, and HeLa cells involving activation of a Src/Shc/ERK signaling pathway and attenuating apoptosis. This action is mediated by the ligand binding domain and eliminated by nuclear targeting of the receptor protein; ERalpha, ERbeta, or AR can transmit it with similar efficiency irrespective of whether the ligand is an estrogen or an androgen. This antiapoptotic action can be dissociated from the transcriptional activity of the receptor with synthetic ligands, providing proof of principle for the development of function-specific-as opposed to tissue-selective-and gender-neutral pharmacotherapeutics.