Efficacious bone regeneration could revolutionize the clinical management of bone and musculoskeletal disorders. Although several bone morphogenetic proteins (BMPs) (mostly BMP-2 and BMP-7) have been shown to induce bone formation, it is unclear whether the currently used BMPs represent the most osteogenic ones. Until recently, comprehensive analysis of osteogenic activity of all BMPs has been hampered by the fact that recombinant proteins are either not biologically active or not available for all BMPs. In this study, we used recombinant adenoviruses expressing the 14 types of BMPs (AdBMPs), and demonstrated that, in addition to currently used BMP-2 and BMP-7, BMP-6 and BMP-9 effectively induced orthotopic ossification when either AdBMP-transduced osteoblast progenitors or the viral vectors were injected into the quadriceps of athymic mice. Radiographic and histological evaluation demonstrated that BMP-6 and BMP-9 induced the most robust and mature ossification at multiple time points. BMP-3, a negative regulator of bone formation, was shown to effectively inhibit orthotopic ossification induced by BMP-2, BMP-6, and BMP-7. However, BMP-3 exerted no inhibitory effect on BMP-9-induced bone formation, suggesting that BMP-9 may transduce osteogenic signaling differently. Our findings suggest that BMP-6 and BMP-9 may represent more effective osteogenic factors for bone regeneration.

Reflexes from the carotid body have been implicated in cardiorespiratory disorders associated with chronic intermittent hypoxia (CIH). To investigate whether CIH causes functional and͞or structural plasticity in the carotid body, rats were subjected to 10 days of recurrent hypoxia or normoxia. Acute exposures to 10 episodes of hypoxia evoked long-term facilitation (LTF) of carotid body sensory activity in CIH-conditioned but not in control animals. The magnitude of sensory LTF depended on the length of CIH conditioning and was completely reversible and unique to CIH, because conditioning with a comparable duration of sustained hypoxia was ineffective. Histological analysis revealed no differences in carotid body morphology between control and CIH animals. Previous treatment with superoxide anion (O 2•؊ ) scavenger prevented sensory LTF. In the CIH-conditioned animals, carotid body aconitase enzyme activity decreased compared with controls. These observations suggest that increased generation of reactive oxygen species contribute to sensory LTF. In CIH animals, carotid body complex I activity of the mitochondrial electron transport is inhibited, suggesting mitochondria as one source of O 2•؊ generation. These observations demonstrate that CIH induces a previously uncharacterized form of reactive oxygen species-dependent, reversible, functional plasticity in carotid body sensory activity. The sensory LTF may contribute to persistent reflex activation of sympathetic nerve activity and blood pressure in recurrent apnea patients experiencing CIH.H ypoxia is a pervasive physiological stimulus. Changes in arterial blood oxygen levels are monitored continuously by peripheral chemoreceptors, especially the carotid bodies (reviewed in ref. 1). The ensuing reflexes are critical for maintaining homeostasis during hypoxia. Hypoxia is encountered under many different circumstances. For instance, sojourn to high altitude exposes individuals to sustained hypoxia (SH). On the other hand, people living at sea level experience intermittent hypoxia more often in life than SH. Many pathophysiological situations including recurrent apnea syndromes (central or obstructive sleep apneas), apneas in premature infants, and asthmatic attacks are associated with intermittent hypoxia. Decreases in arterial blood oxygen occur in both types of hypoxia. However, cardiorespiratory systems adapt to chronic SH to maintain adequate oxygen delivery to tissues (reviewed in refs. 2 and 3), whereas chronic intermittent hypoxia (CIH) leads to serious pathophysiological consequences such as hypertension, myocardial infarcts, and even stroke (4, 5).Reflexes from the carotid body have been proposed to play a critical role in ventilatory and circulatory adaptations to chronic SH and are associated with structural changes in the carotid body including hypertrophy of the organ, increased vascularization, and increased number of glomus cells (the putative oxygensensing cells; reviewed in refs. 2 and 3). There is evidence that reflexes arising from the carotid ...

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Chronic intermittent hypoxia (CIH) occurs in patients with sleep apnoea and has adverse effects on multiple physiological functions. Previous studies have shown that reflexes arising from carotid bodies mediate CIH-evoked cardio-respiratory responses, and reactive oxygen species (ROS) play important roles in eliciting systemic responses to CIH. Very little is known about the molecular mechanisms underlying CIH. The transcriptional activator hypoxia-inducible factor-1 (HIF-1) mediates a broad range of cellular and systemic responses to hypoxia, and HIF-1 is activated in cell cultures exposed to IH. In the present study we examined whether CIH activates HIF-1 and if so whether it contributes to cardio-respiratory responses and ROS generation in mice. Experiments were performed on male littermate wild-type (WT) and heterozygous (HET) mice partially deficient in HIF-1α, the O 2 regulated subunit of the HIF-1 complex. Both groups of mice were exposed to either 10 days of CIH (15 s of hypoxia followed by 5 min of normoxia, 9 episodes h −1 , 8 h day −1 ) or to 10 days of 21% O 2 (controls). Carotid body response to hypoxia was augmented, and acute intermittent hypoxia (AIH) induced sensory long-term facilitation (sLTF) of the chemoreceptor activity in CIH-exposed WT mice. In striking contrast, hypoxic sensory response was unaffected and AIH was ineffective in eliciting sLTF in CIH-exposed HET mice. Analysis of cardio-respiratory responses in CIH-exposed WT mice revealed augmented hypoxic ventilatory response, LTF of breathing, elevated blood pressures and increased plasma noradrenaline. In striking contrast these responses were either absent or attenuated in HET mice exposed to CIH. In CIH-exposed WT mice, ROS were elevated and this response was absent in HET mice. Manganese (III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride, a potent scavenger of superoxide, not only prevented CIH-induced increases in ROS but also CIH-evoked HIF-1α up-regulation in WT mice. These results indicate that: (a) HIF-1 activation is critical for eliciting CIH-induced carotid body-mediated cardio-respiratory responses; (b) CIH increases ROS; and (c) the effects of CIH involve complex positive interactions between HIF-1 and ROS.

Osteoblast lineage-specific differentiation of mesenchymal stem cells is a well regulated but poorly understood process. Both bone morphogenetic proteins (BMPs) and Wnt signaling are implicated in regulating osteoblast differentiation and bone formation. Here we analyzed the expression profiles of mesenchymal stem cells stimulated with Wnt3A and osteogenic BMPs, and we identified connective tissue growth factor (CTGF) as a potential target of Wnt and BMP signaling. We confirmed the microarray results, and we demonstrated that CTGF was up-regulated at the early stage of BMP-9 and Wnt3A stimulations and that Wnt3A-regulated CTGF expression was ␤-catenin-dependent. RNA interference-mediated knockdown of CTGF expression significantly diminished BMP-9-induced, but not Wnt3A-induced, osteogenic differentiation, suggesting that Wnt3A may also regulate osteoblast differentiation in a CTGF-independent fashion. However, constitutive expression of CTGF was shown to inhibit both BMP-9-and Wnt3A-induced osteogenic differentiation. Exogenous expression of CTGF was shown to promote cell migration and recruitment of mesenchymal stem cells. Our findings demonstrate that CTGF is up-regulated by Wnt3A and BMP-9 at the early stage of osteogenic differentiation, which may regulate the proliferation and recruitment of osteoprogenitor cells; however, CTGF is down-regulated as the differentiation potential of committed pre-osteoblasts increases, strongly suggesting that tight regulation of CTGF expression may be essential for normal osteoblast differentiation of mesenchymal stem cells.

Gaseous messengers, nitric oxide and carbon monoxide, have been implicated in O 2 sensing by the carotid body, a sensory organ that monitors arterial blood O 2 levels and stimulates breathing in response to hypoxia. We now show that hydrogen sulfide (H 2 S) is a physiologic gasotransmitter of the carotid body, enhancing its sensory response to hypoxia. Glomus cells, the site of O 2 sensing in the carotid body, express cystathionine γ-lyase (CSE), an H 2 Sgenerating enzyme, with hypoxia increasing H 2 S generation in a stimulus-dependent manner. Mice with genetic deletion of CSE display severely impaired carotid body response and ventilatory stimulation to hypoxia, as well as a loss of hypoxia-evoked H 2 S generation. Pharmacologic inhibition of CSE elicits a similar phenotype in mice and rats. Hypoxia-evoked H 2 S generation in the carotid body seems to require interaction of CSE with hemeoxygenase-2, which generates carbon monoxide. CSE is also expressed in neonatal adrenal medullary chromaffin cells of rats and mice whose hypoxia-evoked catecholamine secretion is greatly attenuated by CSE inhibitors and in CSE knockout mice.I n adult mammals, carotid bodies are the sensory organs responsible for monitoring arterial blood O 2 concentrations and relay sensory information to the brainstem neurons associated with regulation of breathing and the cardiovascular system (1). Carotid bodies are comprised mainly of two cell types: glomus (also called type I) and sustanticular (or type II) cells. Glomus cells, of neuronal nature, are considered the main hypoxiasensing cells. The gaseous messengers, carbon monoxide (CO) and nitric oxide (NO), generated by hemeoxygenase-2 (HO-2) and neuronal nitric oxide synthase (nNOS), respectively, physiologically inhibit carotid body activity (2-4). Because HO-2 and nNOS require molecular O 2 for their activity, stimulation of carotid body activity by hypoxia may reflect in part reduced formation of CO and NO (5).Like NO and CO, hydrogen sulfide (H 2 S) is a gasotransmitter physiologically regulating neuronal transmission (6) and vascular tone (7). Cystathionine γ-lyase (CSE) (EC 4.4.1.1) and cystathionine β-synthase (CBS) (4.2.1.22) are the major enzymes associated with generation of endogenous H 2 S (8, 9). CBS is the predominant H 2 S-synthesizing enzyme in the brain, CSE preponderates in the peripheral tissues whose H 2 S levels are reduced 90% in CSE −/− mice (7-10). Given that carotid bodies are peripheral organs and that H 2 S is redox active, we hypothesized that CSE-derived H 2 S plays a role in hypoxic sensing by the carotid body. We examined carotid body response to hypoxia in wild-type (CSE +/+ ) and CSE −/− mice as well as in rats treated with CSE inhibitor. Genetic deletion or pharmacologic inhibition of CSE dramatically impairs hypoxic sensing by the carotid body as well as in neonatal adrenal medullary chromaffin cells (AMC). ResultsLoss of Carotid Body Response to Hypoxia in CSE −/− Mice. CSE immunoreactivity was seen in glomus cells of carotid bodies from CSE +/+ mice...

Bone morphogenetic proteins (BMPs) belong to the TGF-␤ superfamily and play an important role in development and in many cellular processes. We have found that BMP-2, BMP-6, and BMP-9 induce the most potent osteogenic differentiation of mesenchymal stem cells. Expression profiling analysis has revealed that the Inhibitors of DNA binding/differentiation (Id)-1, Id-2, and Id-3 are among the most significantly up-regulated genes upon BMP-2, BMP-6, or BMP-9 stimulation. Here, we sought to determine the functional role of these Id proteins in BMP-induced osteoblast differentiation. We demonstrated that the expression of Id-1, Id-2, and Id-3 genes was significantly induced at the early stage of BMP-9 stimulation and returned to basal levels at 3 days after stimulation. RNA interference-mediated knockdown of Id expression significantly diminished the BMP-9-induced osteogenic differentiation of mesenchymal progenitor cells. Surprisingly, a constitutive overexpression of these Id genes also inhibited osteoblast differentiation initiated by BMP-9. Furthermore, we demonstrated that BMP-9-regulated Id expression is Smad4-dependent. Overexpression of the three Id genes was shown to promote cell proliferation that was coupled with an inhibition of osteogenic differentiation. Thus, our findings suggest that the Id helix-loop-helix proteins may play an important role in promoting the proliferation of early osteoblast progenitor cells and that Id expression must be down-regulated during the terminal differentiation of committed osteoblasts, suggesting that a balanced regulation of Id expression may be critical to BMP-induced osteoblast lineage-specific differentiation of mesenchymal stem cells.Bone formation is a well orchestrated process of osteoblast lineage-specific differentiation (1). During osteogenesis, pluripotent mesenchymal stem cells differentiate into preosteoblasts rather than serve as progenitor cells for myocytes, adipocytes, or chondrocytes. These preosteoblasts then differentiate into mature osteoblasts that deposit the necessary components to form bone matrix and allow subsequent mineralization. Bone morphogenetic proteins (BMPs) 1 play an important role in regulating osteoblast differentiation and subsequent bone formation (2, 3). BMPs belong to the TGF-␤ superfamily and play an important role in development (4, 5). At least 15 types of BMPs have been identified in humans (4, 6 -8). Genetic disruptions of BMPs have resulted in various skeletal and extraskeletal developmental abnormalities (9).BMP signaling activity is initiated by interaction with the heterodimeric complex of two transmembrane serine/threonine kinase receptors, BMPR types I and II (10, 11) The activated receptor kinases phosphorylate the transcription factors Smads 1, 5, or 8. The phosphorylated Smads then form heterodimeric complexes with Smad4 and activate the expression of target genes in concert with other co-activators (12-14).We have conducted a comprehensive analysis of the osteogenic activity of 14 types of human BMPs (i.e. BMP-2 to BM...