The HIF signaling pathway in osteoblasts directly modulates erythropoiesis through the production of EPO.

Osteoblasts are an important component of the hematopoietic microenvironment in bone. However, the mechanisms by which osteoblasts control hematopoiesis remain unknown. We show that augmented HIF signaling in osteoprogenitors results in HSC niche expansion associated with selective expansion of the erythroid lineage. Increased red blood cell production occurred in an EPO-dependent manner with increased EPO expression in bone and suppressed EPO expression in the kidney. In contrast, inactivation of HIF in osteoprogenitors reduced EPO expression in bone. Importantly, augmented HIF activity in osteoprogenitors protected mice from stress-induced anemia. Pharmacologic or genetic inhibition of prolyl hydroxylases1/2/3 in osteoprogenitors elevated EPO expression in bone and increased hematocrit. These data reveal an unexpected role for osteoblasts in the production of EPO and modulation of erythropoiesis. Furthermore, these studies demonstrate a molecular role for osteoblastic PHD/VHL/HIF signaling that can be targeted to elevate both HSCs and erythroid progenitors in the local hematopoietic microenvironment.

Severe Extracellular Matrix Abnormalities and Chondrodysplasia in Mice Lacking Collagen Prolyl 4-Hydroxylase Isoenzyme II in Combination with a Reduced Amount of Isoenzyme I.

Collagen prolyl 4-hydroxylases (C-P4H-I, C-P4H-II, and C-P4H-III) catalyze formation of 4-hydroxyproline residues required to form triple-helical collagen molecules. Vertebrate C-P4Hs are α2ß2 tetramers differing in their catalytic α subunits. C-P4H-I is the major isoenzyme in most cells, and inactivation of its catalytic subunit (P4ha1(-/-)) leads to embryonic lethality in mouse, whereas P4ha1(+/-) mice have no abnormalities. To study the role of C-P4H-II, which predominates in chondrocytes, we generated P4ha2(-/-) mice. Surprisingly, they had no apparent phenotypic abnormalities. To assess possible functional complementarity, we established P4ha1(+/-);P4ha2(-/-) mice. They were smaller than their littermates, had moderate chondrodysplasia, and developed kyphosis. A transient inner cell death phenotype was detected in their developing growth plates. The columnar arrangement of proliferative chondrocytes was impaired, the amount of 4-hydroxyproline and the Tm of collagen II were reduced, and the extracellular matrix was softer in the growth plates of newborn P4ha1(+/-);P4ha2(-/-) mice. No signs of uncompensated ER stress were detected in the mutant growth plate chondrocytes. Some of these defects were also found in P4ha2(-/-) mice, although in a much milder form. Our data show that C-P4H-I can to a large extent compensate for the lack of C-P4H-II in proper endochondral bone development, but their combined partial and complete inactivation, respectively, leads to biomechanically impaired extracellular matrix, moderate chondrodysplasia, and kyphosis. Our mouse data suggest that inactivating mutations in human P4HA2 are not likely to lead to skeletal disorders, and a simultaneous decrease in P4HA1 function would most probably be required to generate such a disease phenotype.

Fibrosis and hypoxia-inducible factor-1α-dependent tumors of the soft tissue on loss of von Hippel-Lindau in mesenchymal progenitors.

The hypoxia-inducible factor (Hif)-1α (Hif-1α) and Hif-2α (Epas1) have a critical role in both normal development and cancer. von Hippel Lindau (Vhl) protein, encoded by a tumor suppressor gene, is an E3 ubiquitin ligase that targets Hif-1α and Epas1 to the proteasome for degradation. To better understand the role of Vhl in the biology of mesenchymal cells, we analyzed mutant mice lacking Vhl in mesenchymal progenitors that give rise to the soft tissues that form and surround synovial joints. Loss of Vhl in mesenchymal progenitors of the limb bud caused severe fibrosis of the synovial joints and formation of aggressive masses with histologic features of mesenchymal tumors. Hif-1α and its downstream target connective tissue growth factor were necessary for the development of these tumors, which conversely still developed in the absence of Epas1, but at lower frequency. Human tumors of the soft tissue are a very complex and heterogeneous group of neoplasias. Our novel findings in genetically altered mice suggest that activation of the HIF signaling pathway could be an important pathogenetic event in the development and progression of at least a subset of these tumors.

Is periodontitis an independent risk factor for subclinical atherosclerosis?

OBJECTIVES: The aim of this study was to assess the interrelationship between periodontitis and atherosclerosis by comparing the ultrasound and clinical markers of atherosclerosis in systemically healthy patients with and without periodontitis and whether periodontitis can be an independent risk factor for atherosclerosis. MATERIALS AND METHODS: Total 40 subjects, of same socioeconomic status, belonging to age group of 35-65 years, were recruited and divided into two groups - Group I (Chronic Generalised Periodontitis without any systemic disease: CP-SH), Group II (Normal healthy patients without periodontitis and any systemic disease - SH). Clinical measurements and ultrasound examinations were carried out. Qualitative variables were analyzed using Chi square test and qualitative variables using Unpaired Student t test. Statistical significance was accepted for p≤0.05. RESULTS: Carotid ultrasound revealed right and left intima media thickness (IMT) of 0.626±0.016mm and 0.715±0.037mm respectively in cases versus 0.495±0.009mm and 0.518±0.009mm respectively in controls, with the difference being statistically significant. In cases, mean diastolic blood pressure (DBP) was 83.45±4.07mmHg versus 79.25±3.63mmHg in controls, with the difference being statistically significant. CONCLUSION: In this study, we found statistically significant differences in carotid IMT and DBP values between cases and controls. These findings suggest independent role of periodontal disease in subclinical atherosclerosis.

Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development.

Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.

Hypoxia-inducible factor-1 (HIF-1) but not HIF-2 is essential for hypoxic induction of collagen prolyl 4-hydroxylases in primary newborn mouse epiphyseal growth plate chondrocytes.

Hypoxia-inducible factors (HIFs) are the master regulators of hypoxia-responsive genes. They play a critical role in the survival, development, and differentiation of chondrocytes in the avascular hypoxic fetal growth plate, which is rich in extracellular matrix (ECM) and in its main component, collagens. Several genes involved in the synthesis, maintenance, and degradation of ECM are regulated by HIFs. Collagen prolyl 4-hydroxylases (C-P4Hs) are key enzymes in collagen synthesis because the resulting 4-hydroxyprolines are necessary for the stability of all collagen molecules. The vertebrate C-P4Hs are α(2)ß(2) tetramers with three isoforms of the catalytic α subunit, yielding C-P4Hs of types I-III. C-P4H-I is the main form in most cells, but C-P4H-II is the major form in chondrocytes. We postulated here that post-translational modification of collagens, particularly 4-hydroxylation of proline residues, could be one of the modalities by which HIF regulates the adaptive responses of chondrocytes in fetal growth plates. To address this hypothesis, we used primary epiphyseal growth plate chondrocytes isolated from newborn mice with conditionally inactivated genes for HIF-1α, HIF-2α, or the von Hippel-Lindau protein. The data obtained showed that C-P4H α(I) and α(II) mRNA levels were increased in hypoxic chondrocytes in a manner dependent on HIF-1 but not on HIF-2. Furthermore, the increases in the C-P4H mRNA levels were associated with both increased amounts of the C-P4H tetramers and augmented C-P4H activity in hypoxia. The hypoxia inducibility of the C-P4H isoenzymes is thus likely to ensure sufficient C-P4H activity for collagen synthesis occurring in chondrocytes in a hypoxic environment.

A novel population of cells expressing both hematopoietic and mesenchymal markers is present in the normal adult bone marrow and is augmented in a murine model of marrow fibrosis.

Bone marrow (BM) fibrosis is a feature of severe hyperparathyroidism. Consistent with this observation, mice expressing constitutively active parathyroid hormone (PTH)/PTH-related peptide receptors (PPR) in osteoblasts (PPR*Tg) display BM fibrosis. To obtain insight into the nature of BM fibrosis in such a model, a double-mutant mouse expressing constitutively active PPR and green fluorescent protein (GFP) under the control of the type I collagen promoter (PPR*Tg/GFP) was generated. Confocal microscopy and flow cytometry revealed the presence of a cell population expressing GFP (GFP(+)) that was also positive for the hematopoietic marker CD45 in the BM of both PPR*Tg/GFP and control animals. This cell population was expanded in PPR*Tg/GFP. The existence of cells expressing both type I collagen and CD45 in the adult BM was confirmed by IHC and fluorescence-activated cell sorting. An analysis of total RNA extracted from sorted GFP(+)CD45(+) cells showed that these cells produced type I collagen and PTH/PTH-related peptide receptor and receptor activator for NF-κB mRNAs, further supporting their features of being both mesenchymal and hematopoietic lineages. Similar cells, known as fibrocytes, are also present in pathological fibroses. Our findings, thus, indicate that the BM is a permissive microenvironment for the differentiation of fibrocyte-like cells and raise the possibility that these cells could contribute to the pathogenesis of BM fibrosis.

Osteoprotegerin abrogated cortical porosity and bone marrow fibrosis in a mouse model of constitutive activation of the PTH/PTHrP receptor.

Intracortical porosities and marrow fibrosis are hallmarks of hyperparathyroidism and are present in bones of transgenic mice expressing constitutively active parathyroid hormone/parathyroid hormone-related protein receptors (PPR*Tg). Cortical porosity is the result of osteoclast activity; however, the etiology of marrow fibrosis is poorly understood. While osteoclast numbers and activity are regulated by osteoprotegerin (OPG), bisphosphonates suppress osteoclast activity but not osteoclast numbers. We therefore used OPG and bisphosphonates to evaluate the extent to which osteoclasts, as opposed to bone resorption, regulate marrow fibrosis in PPR*Tg mice after treatment of animals with vehicle, OPG, alendronate, or zoledronate. All three agents similarly increased trabecular bone volume in both PPR*Tg and control mice, suggesting that trabecular bone resorption was comparably suppressed by these agents. However, the number of trabecular osteoclasts was greatly decreased by OPG but not by either alendronate or zoledronate. Furthermore, intracortical porosity and marrow fibrosis were virtually abolished by OPG treatment, whereas alendronate and zoledronate only partially reduced these two parameters. The greater reductions in cortical porosity and increments in cortical bone mineral density with OPG in PPR*Tg mice were associated with greater improvements in bone strength. The differential effect of OPG versus bisphosphonates on marrow fibrosis, despite similar effects on trabecular bone volume, suggests that marrow fibrosis was related not only to bone resorption but also to the presence of osteoclasts.

Global Perspectives in Controversies Related to the Management of Intra-Abdominal Infections.

Background: Intra-abdominal infections (IAI) remain a substantial cause of worldwide morbidity, mortality, and healthcare cost burden. The World Surgical Infection Society (WSIS) was organized to help improve global outcomes from surgical infections. An initial project for the WSIS was to assess how surgeons treat common IAI in their regions. Methods: A 10-item questionnaire was distributed to members of four surgical societies dedicated to the study of surgical infections. Questions were related to common treatment decisions in the management of IAI, with the intention of identifying differences and potential controversies in patient care. Responses were analyzed by comparing percentages with 95% confidence intervals. Results: Globally, management was relatively similar for peritoneal irrigation (most commonly with saline or other crystalloid: China, 83.2% ± 5.8%; North America, 93.2% ± 6.4%; Europe, 85.7% ± 25.9%; and Latin America, 71.8% ± 6.9%). More varied responses were seen for the management of specific disease states; for instance, for cholangitis, endoscopic retrograde cholangiopancreatic interventions were more common in North America (83.1% ± 9.6%) and less common in China (28.1% ± 7.0%). For appendiceal abscesses, percutaneous drainage and antibiotic treatment was most common in North America (93.2% ± 6.4%) and least common in Latin America (19.6% ± 6.1%). Additionally, the management of fascial and wound closures were different by region. Vacuum-assisted wound closure after fascial closure was utilized commonly in North America (32.2% ± 11.9%), Europe (28.6% ± 33.5%), and Latin America (27.6% ± 6.9%), however, was less commonly utilized in China (9.9% ± 4.4%), where there was higher rate of primary skin closure (85.7% ± 5.4%). Conclusion: Through its partnership with other surgical infection societies, the WSIS aims to develop evidence-based guidelines for more consistent pattern of IAI management globally. Delving further into why their practices differ may help improve worldwide outcomes.

Short term outcomes and unintended benefits of establishing a HPB program at a university-affiliated community hospital.

BACKGROUND: In hepato-pancreato-biliary (HPB) surgery higher volumes are associated with improved outcomes; however, there are limitations to regionalization. Here we report our experience establishing multidisciplinary HPB program at a university-affiliated community hospital. METHODS: This is a retrospective review of patients who underwent HPB surgery between 2015 and 2017. Chief residents' HPB case logs were collected. RESULTS: 61 pancreatic resections and 62 hepatic resections were performed. The morbidity, 30-day mortality and median length of stay following pancreatic resections were 27%, 1.5%, and 8 days, respectively. The morbidity, 90-day mortality, and median length of stay following hepatic resections were 24%, 3%, and 7 days, respectively. The median pancreatic and liver case volumes for graduating chief residents increased from 7 to 8 to 16 and 16, respectively (p < 0.05), after the establishment of a HPB program. Participation in multidisciplinary care (p = 0.08) and clinical trial enrollment increased. CONCLUSION: Our study demonstrates short-term outcomes comparable to high volume centers. Development of a HPB program had a positive impact on resident operative experience, increased multidisciplinary care and increased clinical trial enrollment.

Merge to emerge - An interdisciplinary approach for management of periodontally compromised orthodontically treated patient.

In the modern day dental practice, synergy is fundamental. This synergistic effect must exist among various disciplines of dentistry for proper diagnosis, treatment planning, sequencing and execution of treatment in complex and challenging dental situations. Such collaborative effect between an orthodontist and a periodontist is essential as both works with same element, the tooth as crown-root unit with its supporting tissues. The orthodontic treatment is carried out through the medium of periodontium, so a healthy tooth supporting system is an essential prerequisite. Every potential candidate for orthodontic treatment should undergo a thorough periodontal examination. Any lousily diagnosed or conducted orthodontic treatment could be a facilitator of periodontal inflammatory or infectious process especially when the patient's oral hygiene is explicitly deficient. This case report demonstrates a challenging situation to a periodontist where patient had completed her orthodontic treatment but ended up with severe periodontal disease. The patient was thoroughly examined and a comprehensive treatment was planned and executed. Regenerative surgical procedures were done using platelet rich fibrin and hydroxyapitatite bone graft. Patient was followed up for 2 years. As sequelae of surgical procedures, patient had developed black triangles in the anterior region. The patient was unwilling for further perioplastic surgical procedures and further orthodontic treatment, so a gingival prosthesis using valplast was fabricated addressing her esthetic concerns.

Frenectomy with semilunar coronally repositioned flap: A single stage approach - simple solution for complex problem.

Gingival recession is defined as the displacement of gingival margin apical to cementoenamel junction. Aberrant frenum attachment can contribute to the progression of recession by generating tension on the marginal tissues. Treating such defects is a two stage procedure-frenectomy and recession coverage procedure. New techniques are developed to increase the predictability, reduce patient discomfort and number of surgical sites. Also, these techniques try to satisfy patients esthetic demands, which include the final colour and tissue blend of the covered area. In this case report, we present a method for coronally repositioning gingiva for root coverage over the maxillary central incisors while simultaneously performing a frenectomy, thus being clinically advantageous compared to two-stage technique.

From hopeless to good prognosis: journey of a failing tooth.

Chronic periodontitis, along with associated clinical findings such as pathologic tooth migration, diastema, functional and aesthetic aberrations, poses an immense challenge to a dental professional. These findings convert clinical decision making into a daunting task and adversely affect the prognosis and the treatment plan for the presenting clinical problem. An interdisciplinary approach aimed at restoring functional and aesthetic needs of the affected individual within the limitations of such a compromised clinical scenario may be a viable alternative to any radical treatment causing loss of natural tooth structure such as extraction. This article reports the usefulness of the interdisciplinary route for managing an otherwise hopeless clinical situation of chronic periodontitis complicated with extreme mobility and pathologic tooth migration, which resulted in compromised function and aesthetics.

Vertical and horizontal ridge augmentation in anterior maxilla using autograft, xenograft and titanium mesh with simultaneous placement of endosseous implants.

Endosseous implants have revolutionized the field of Implants and Periodontics. Implant placement is a viable option in the treatment of partial and full edentulism. However, placement of implants in alveolar deficiencies may lead to adverse angulations, mechanical overload and esthetic dissatisfaction. When minimum dimensions for implant placement are not present in alveolar process, it is necessary to augment the size of the ridge. This can be achieved by various methods and materials. Here we present a successful case of vertical and horizontal ridge augmentation in anterior maxilla using autograft, xenograft and titanium mesh with simultaneous placement of implants, where autograft was obtained from the same site avoiding secondary surgical site.

VEGF-independent cell-autonomous functions of HIF-1α regulating oxygen consumption in fetal cartilage are critical for chondrocyte survival.

Fetal growth plate cartilage is nonvascularized, and chondrocytes largely develop in hypoxic conditions. We previously found that mice lacking the hypoxia-inducible transcription factor HIF-1α in cartilage show massive death of centrally located, hypoxic chondrocytes. A similar phenotype was observed in mice with genetic ablation of either all or specifically the diffusible isoforms of vascular endothelial growth factor (VEGF), a prime angiogenic target of HIF-1α. Here, we assessed whether VEGF is a critical downstream component of the HIF-1α-dependent survival pathway in chondrocytes. We used a genetic approach to conditionally overexpress VEGF164 in chondrocytes lacking HIF-1α, evaluating potential rescuing effects. The effectiveness of the strategy was validated by showing that transgenic expression of VEGF164 in Col2-Cre;VEGF(f/f) mice stimulated angiogenesis in the perichondrium, fully corrected the excessive hypoxia of VEGF-deficient chondrocytes, and completely prevented chondrocyte death. Yet, similarly crossed double-mutant embryos lacking HIF-1α and overexpressing VEGF164 in the growth plate cartilage still displayed a central cell death phenotype, albeit slightly delayed and less severe compared with mice exclusively lacking HIF-1α. Transgenic VEGF164 induced massive angiogenesis in the perichondrium, yet this only partially relieved the aberrant hypoxia present in HIF-1α-deficient cartilage and thereby likely inflicted only a partial rescue effect. In fact, excessive hypoxia and failure to upregulate phosphoglycerate-kinase 1 (PGK1), a key enzyme of anaerobic glycolytic metabolism, were among the earliest manifestations of HIF-1α deficiency in cartilaginous bone templates, and reduced PGK1 expression was irrespective of transgenic VEGF164. These findings suggest that HIF-1α activates VEGF-independent cell-autonomous mechanisms to sustain oxygen levels in the challenged avascular cartilage by reducing oxygen consumption. Hence, regulation of the metabolic pathways by HIF-1α and VEGF-dependent regulation of angiogenesis coordinately act to maintain physiological cartilage oxygenation. We conclude that VEGF and HIF-1α are critical preservers of chondrocyte survival by ensuring an adequate balance between availability and handling of oxygen in developing growth cartilage.

Role of hypoxia-inducible factor-1alpha in angiogenic-osteogenic coupling.

Angiogenesis and osteogenesis are tightly coupled during bone development and regeneration. The vasculature supplies oxygen to developing and regenerating bone and also delivers critical signals to the stroma that stimulate mesenchymal cell specification to promote bone formation. Recent studies suggest that the hypoxia-inducible factors (HIFs) are required for the initiation of the angiogenic-osteogenic cascade. Genetic manipulation of individual components of the HIF/vascular endothelial growth factor (VEGF) pathway in mice has provided clues to how coupling is achieved. In this article, we review the current understanding of the cellular and molecular mechanisms responsible for angiogenic-osteogenic coupling. We also briefly discuss the therapeutic manipulation of HIF and VEGF in skeletal repair. Such discoveries suggest promising approaches for the development of novel therapies to improve bone accretion and repair.

About the importance of being desulfated.

Sulfated proteoglycans have important structural and signaling functions in the growth plate. In the October 1, 2008, issue of Genes & Development, Settembre and colleagues (2645-2650) report that lack of SUMF1, a crucial enzyme in the activation of sulfatases, causes a severe chondrodysplasia by augmenting fibroblast growth factor signaling and by hampering the autophagic process, which the investigators show is constitutively on in chondrocytes. The findings highlight the essential role of desulfation in cartilage biology and organogenesis.