Nitric oxide synthase isoforms during fracture healing.

We have shown previously that nitric oxide (NO) has regulatory effects on fracture healing. Our aim here was to investigate the temporal expression patterns of the three NO synthase (NOS) isoforms that are responsible for the generation of NO by semiquantitative competitive polymerase chain reaction (PCR) and immunoblot analysis after femoral fractures in rats. We found that 4 days after fracture, there were increases in the levels of messenger RNA (mRNA) for all three NOS isoforms, with peaks for the inducible NOS (iNOS; 35-fold increase, p < 0.05) at day 4, the endothelial NOS (eNOS; 5-fold increase, p < 0.05) at day 7, and the neuronal NOS (bNOS; 16-fold increase, p < 0.05) at day 21. At a protein level, the time course expression of NOS isoforms was consistent with the results of those at the mRNA level. In addition, we have previously reported a 2.5-fold increase in NOS activity detected by [3H]arginine to [3H]citrulline conversion at day 15 compared with that at day 4 after fracture. The findings that the expression of NOS isoforms during fracture healing is type specific and time dependent are important and may have clinical applications in the regulation of bone repair by NOS inhibitors or stimulators at different stages after injury.     

Nitric oxide modulates water and electrolyte transport in the ileum.

OBJECTIVE: To test the hypothesis that nitric oxide is a modulator of ileal water and ion transport. SUMMARY BACKGROUND DATA: Nitric oxide is produced in the vascular endothelium and enteric neural plexuses of the intestine and is involved in gastrointestinal motility and smooth muscle contractility. Little is known about the role of nitric oxide in intestinal epithelial transport. METHODS: Ten-centimeter rabbit ileal segments (n = 50) were vascularly perfused with an electrolyte solution containing red cells. The lumen was perfused with a solution containing 14C-PEG. Net fluxes of water and ions were calculated during three 20-minute periods: basal, drug infusion, and recovery. Perfusion pressure was recorded to document changes in vascular resistance. Agents infused included the nitric oxide synthase substrate L-arginine, the nitric oxide source sodium nitroprusside, the substrate control D-arginine, and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester. RESULTS: L-arginine and sodium nitroprusside caused absorption of water and ions. NG-nitro-L-arginine methyl ester caused secretion of water and ions, which was prevented by synchronous infusion of L-arginine. Infusion of D-arginine had no effect. Both L-arginine and sodium nitroprusside caused mild vasodilation. CONCLUSIONS: Inhibition of endogenous nitric oxide synthesis by NG-nitro-L-arginine methyl ester causes secretion of water and ions. This secretion is reversed by administration of the nitric oxide synthase substrate L-arginine. These findings are consistent with the hypothesis that endogenous nitric oxide has a proabsorptive influence over the ileum in the basal state.     

Localization of nitric oxide synthases during fracture healing.

Previously, we have reported that nitric oxide synthases (NOSs), which generate NO, modulate fracture healing. However, the cellular sources of the NOS isoforms during the course of fracture healing have not been studied systematically. The purpose of this study was to localize the cellular distribution of NOS isoforms (inducible NOS [iNOS], endothelial NOS [eNOS], and neuronal NOS [bNOS]) by in situ hybridization and immunohistology after femoral fractures in rats. The iNOS signal was detected during the initial stages (on day 4 and day 7) of fracture healing in 52 +/- 2% (mean +/- SE, n = 7) of cells within the intramembranous region, along the edge of the periosteal callus. The iNOS signal in callus cells declined to an undetectable level on day 14. eNOS was detected during the middle stages (on day 7 and day 14) of fracture healing in cells lining the blood vessels and also in 49 +/- 3% of cells in the chondral region. The bNOS signal was found to be increased at the later stages (day 14 and day 21) of fracture healing in 51 +/- 3% of cells at the junction between fibrous tissue and cartilage within the fibrochondral region. In summary, the,expression of NOS isoforms during fracture healing was time dependent and cellular distinctive.     

Nitric oxide enhances experimental wound healing in diabetes

BACKGROUND: Diabetes is characterized by a nitric oxide deficiency at the wound site. This study investigated whether exogenous nitric oxide supplementation with the nitric oxide donor molsidomine (N-ethoxycarbomyl-3-morpholinyl-sidnonimine) could reverse the impaired healing in diabetes. METHODS: Wound healing was studied by creating a dorsal skin incision with subcutaneous polyvinyl alcohol sponge implantation in diabetic and non-diabetic rats. Half of each group was treated with molsidomine. Collagen metabolism was assessed by wound breaking strength, hydroxyproline (OHP) content, RNA expression for collagen type I and III, and matrix metalloproteinase (MMP) 2 activity in wound sponges. Wound fluid, plasma and urinary nitric oxide metabolite levels, and the number of inflammatory cells were assessed. RESULTS: OHP content and wound breaking strength were significantly increased by molsidomine. MMP-2 activity in wound fluid was decreased in diabetes and upregulated by nitric oxide donors. The impaired inflammatory reaction in diabetes was unaffected by nitric oxide donor treatment and ex vivo nitric oxide synthesis was no different between wound macrophages from control and diabetic animals, suggesting that the nitric oxide deficiency in the wound is due to a smaller inflammatory reaction in diabetes. CONCLUSION: The nitric oxide donor molsidomine can at least partially reverse impaired healing associated with diabetes.     

Nitric oxide mediated vasoreactivity during fracture repair

An experimental model of fracture healing has been used to investigate whether nitric oxide mediated vascular reactivity, determined using laser Doppler flowmetry, is present in bone after a fracture. Times corresponding to Days 0, 1, 3, 7, 14, and 28 after fracture were used to study the injured and contralateral limbs in response to bolus intravenous administration of nitric oxide inhibitor, N-nitro-L-arginine methyl ester, and nitric oxide stimulator, acetylcholine. N-nitro-L-arginine methyl ester administration (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) caused a dose dependent increase in systemic blood pressure in each of the assessment groups; however, there was no statistical difference between the groups. Doppler flow readings at the fracture site showed measurable changes in local vascular reactivity after drug administration. At Day 1 after fracture, the magnitude of unit change in vascular reactivity in response to N-nitro-L-arginine methyl ester (1 mumol/kg, 10 mumol/kg, and 100 mumol/kg) was significantly higher in the fractured limb compared with the contralateral limb and also when compared with other points of assessment. These results show that nitric oxide mediated vasoreactivity is present about a fracture site and is maximal in the early healing phase, before returning to basal levels as healing progresses. This is compatible with an initial restoration of blood flow at a fracture site by nitric oxide dependent vasodilation of preexisting blood vessels, followed by ingrowth of less nitric oxide dependent angiogenic vessels during the later phase of repair.     

Nitric oxide signaling modulates synaptic transmission during early postnatal development

Early γ-aminobutyric acid mediated (GABAergic) synaptic transmission and correlated neuronal activity are fundamental to network formation; however, their regulation during early postnatal development is poorly understood. Nitric oxide (NO) is an important retrograde messenger at glutamatergic synapses, and it was recently shown to play an important role also at GABAergic synapses in the adult brain. The subcellular localization and network effect of this signaling pathway during early development are so far unexplored, but its disruption at this early age is known to lead to profound morphological and functional alterations. Here, we provide functional evidence--using whole-cell recording--that NO signaling modulates not only glutamatergic but also GABAergic synaptic transmission in the mouse hippocampus during the early postnatal period. We identified the precise subcellular localization of key elements of the underlying molecular cascade using immunohistochemistry at the light--and electron microscopic levels. As predicted by these morpho-functional data, multineuron calcium imaging in acute slices revealed that this NO-signaling machinery is involved also in the control of synchronous network activity patterns. We suggest that the retrograde NO-signaling system is ideally suited to fulfill a general presynaptic regulatory role and may effectively fine-tune network activity during early postnatal development, while GABAergic transmission is still depolarizing.     

Neuropeptide Y modulates fracture healing through Y1 receptor signaling

Neuropeptide Y acting via it's Y₁ receptor represents a powerful pathway in the control of bone mass. The global or osteoblast‐specific Y₁ receptor deletion induces pronounced bone anabolic effects in mice. However, the contribution of Y₁ receptor deletion in bone repair/healing remained to be clarified. Therefore, in this study we characterized the role of Y₁ receptor deletion in fracture healing. Closed tibial fractures were generated in germline (Y₁^?/?) and osteoblastic‐specific Y₁ receptor knockout mice. The progression of tibial repair monitored from 1‐ until 6‐weeks post‐fracture demonstrated that in Y₁^?/? mice there is a delay in fracture repair, as seen by a decrease in bone callus volume and callus strength. Moreover, the histological features included elevated avascular and cartilage area and consequently delayed cartilage removal, and hence impaired union. Interestingly, this delay in bone repair was not related directly to Y₁ receptors expressed by mature osteoblasts. These findings suggest that the global absence of the Y₁ receptor delays fracture healing, through impairing the early phases of fracture repair to achieve bony union. The data acquired on the role of Y₁ receptor signaling disruption in bone regeneration is critical for the design of future therapeutic strategies. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:1570–1578, 2013

     

一氧化氮调节骨折愈合的实验研究

[目的] 探讨一氧化氮在骨折愈合中的可能作用。[方法] Wistar大鼠60只随机分为实验组和对照组。两组动物的饮用水分别含L-硝基-精氨酸甲基脂（L-NAME）和D-硝基-精氨酸甲基脂（D-NAME），建立股骨开放骨折模型，骨折后第3d、1、2和4周，取每组6只大鼠骨折处骨痂行HE染色观察。另6只大鼠于骨折后4周行骨痂的面积、骨密度和骨折的生物力学评价。[结果] 骨折后两组大鼠体重增加无差别。HE染色光镜下见骨折后不同时期L-NAME组骨折处骨痂生成少，骨痂塑形慢，未成熟细胞比例较对照组多（P〈0．05），加入L-NAME后使骨折愈合反应减小，实验组骨痂的大小、骨密度和愈合强度均明显低于对照组，有明显的统计学意义。[结论] 在骨折愈合过程中加入L-NAME会使骨折愈合减慢，一氧化氮可能促进骨折的愈合过程。     

Nitric oxide in fracture repair. Differential localisation, expression and activity of nitric oxide synthases

Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.     

Nitric oxide levels in wound fluid may reflect the healing trajectory

We analyzed nitric oxide metabolites (nitrate and nitrite, NOx) and other biomarkers in human wound fluids and correlated these markers with wound healing status (progressing or worsening) based on patient's wound history. Samples were collected pre‐ and postcleansing from patients with wounds of various etiologies and analyzed for NOx, matrix metalloproteinase activity, and elastase activity. A laboratory method was developed to analyze NOx which can detect at least 5 μM in samples as small as 10 μL. A nitrate‐free sample collection device was identified to match the sensitivity of this new assay (most “nitrate‐free” products tested contained nitrate levels higher than this detection limit when extracted in such a small volume). The correlation between pre‐ and postcleansing biomarker values, and the diagnostic potential of the biomarkers to wound progress were analyzed. Fifty wounds provided samples that were suitable for NOx analysis. The pre‐ and postcleansing values for NOx showed good correlation (r = 0.72); the correlation was not very strong for matrix metalloproteinase and elastase. Data analysis showed that NOx represents the best metabolite to discriminate between worsening and progressing wounds, and suggested that a two cut point diagnostic test using NOx is better than a single cut point test to identify progressing from worsening wounds.     

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells

Nitric oxide modulates expression of matrix metalloproteinase-9 in rat mesangial cells. BACKGROUND: High-output levels of nitric oxide (NO) are produced by rat mesangial cells (MCs) in response to proinflammatory cytokines such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) by the inducible isoform of NO synthase (iNOS). We tested modulatory effects of NO on the expression and activities of matrix metalloproteinases-9 and -2 (MMP-9 and MMP-2), respectively. Temporal and spatial expression of these MMPs and their specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), seems to be critical in the extensive extracellular matrix (ECM) remodeling that accompanies sclerotic processes of the mesangium. Methods and Results. Using the NO donors S-Nitroso-N-acetyl-D,L-penicillamine (SNAP) and DETA-NONOate, we found strong inhibitory effects of NO mainly on the IL-1beta-induced MMP-9 mRNA levels. NO on its own had only weak effects on the expression of MMP-9 and MMP-2. The addition of the NOS inhibitor NG-monomethyl L-arginine (L-NMMA) dose dependently increased steady-state mRNA levels of cytokine-induced MMP-9, suggesting that endogenously produced NO exerts tonic inhibition of MMP-9 expression. MMP-9 activity in conditioned media from MCs costimulated with IL-1beta and NO donor contained less gelatinolytic activity than media of cells treated with IL-1beta alone. Exogenously added NO did not alter gelatinolytic activity of MMP-9 in cell-free zymographs. The expression levels of TIMP-1 were affected by NO similarly to the expression of MMP-9. CONCLUSION: We conclude that NO modulates cytokine-mediated expression of MMP-9 and TIMP-1 in rat MCs in culture. Our results provide evidence that NO-mediated attenuation of MMP-9 gelatinolytic activity is primarily due to a reduced expression of MMP-9 mRNA, and not the result of direct inhibition of enzymatic activity.     

Nitric oxide modulates renal vasoconstrictor effect of endothelin-1 in conscious lambs

To test the hypothesis that nitric oxide (NO) buffers the renal vasoconstrictor effects of endothelin-1 (ET-1) early in life, renal haemodynamic responses to ET-1 were measured in the presence and absence of endogenously produced NO in conscious lambs. Renal haemodynamic effects of ET-1 were measured for 5 min before (control) and 20 min after intraarterial injection of ET-1 before and after pretreatment with 20 mg/kg of the l-arginine analogue N^G-nitro-l-arginine methyl ester (l-NAME), (experiment 1) and its inactive isomer D-NAME (experiment 2) in conscious lambs aged ~1 week (N=7) and ~6 weeks (N=6). The two experiments were carried out in random order at intervals of 24–48 h. In lambs aged ~6 weeks, a marked increase in renal vascular resistance (RVR) was elicited by ET-1 administration; this response was enhanced twofold following pretreatment with l-NAME. In 1-week-old lambs, however, an increase in RVR in response to ET-1 occurred only after pretreatment with l-NAME. Therefore, we accept our hypothesis and conclude that NO buffers the renal vasoconstrictor effects of ET-1 early in life.The current address of Dr. Liesbeth van der Velde is UCLA Medical Center, Los Angeles, CA, USA. The current address of Dr. Alp Sener is University of Western Ontario, London, ON, Canada     

Nitric oxide in rheumatology.

Nitric oxide (NO) is attracting considerable interest because it mediates many functions. This gas is ubiquitously produced in the body by three enzymes, called NO synthases. Two NO synthases are constitutively expressed, one in the nervous system and the other in the blood vessels, where it regulates tissue perfusion. The third NO synthase can be induced by several stimuli (bacterial endotoxins, cytokines), most notably in inflammatory cells and chondrocytes. The effects of NO produced by the inducible NO synthase range from T-cell response modulation to formation of free radicals responsible fortissue damage and cartilage matrix degradation. Administration of NO synthase inhibitors in animal models of arthritis yields ambiguous effects, often with prevention of arthritis, but sometimes with worsening of established arthritis. The data available to date do not support the use of such inhibitors in the treatment of human arthritis.     

Nitric oxide in osteoarthritis.

Activated articular chondrocytes produce large amounts of nitric oxide (NO), and there is increasing evidence that this is involved in the etiopathogenesis of osteoarthritis (OA). Because of its short half-life, the biological effects of endogenously produced NO are likely to occur locally within the cartilage. We have observed that inhibitors of NO synthases relieve the inhibition of matrix synthesis that otherwise occurs in response to IL-1. To avoid the use of inhibitors, we have recently transduced chondrocytes with the iNOS (NOS-2) gene and confirmed the ability of the endogenously produced NO to inhibit matrix synthesis. Despite the high levels of NO made by these cells, there was no evidence of apoptosis or other forms of cell death. NO was also shown to inhibit the production of TGF-beta(1)by cells treated with IL-1, as well as to decrease matrix production in response to IGF-1. The hypothesis that NO inhibits matrix production by interfering with important autocrine and paracrine factors should be entertained.     

Nitric oxide modulates vascular endothelial growth factor and receptors in chronic cyclosporine nephrotoxicity

BACKGROUND: Vascular endothelial growth factor (VEGF) is involved in angiogenesis, wound healing, and inflammation and exerts its effect via tyrosine kinase receptors, fms-like tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1 or KDR). We have previously shown that VEGF is up-regulated in a model of chronic cyclosporine (CsA) nephrotoxicity and that l-arginine (l-Arg) improved while N-nitro-l-arginine-methyl ester (L-NAME) worsened fibrosis. We examined the role of nitric oxide modulation on VEGF in this model. METHODS: Pair-fed salt-depleted rats were administered CsA, CsA + L-NAME, CsA +l-Arg, vehicle (VH), VH + L-NAME or VH +l-Arg and were sacrificed at 7 or 28 days. Physiologic and histologic changes were studied in addition to the mRNA expression of VEGF and its receptors Flt-1 and KDR/Flk-1 by Northern blot and the protein expression of VEGF by Western blot and immunohistochemical staining. RESULTS: While L-NAME worsened renal function and histology, l-Arg had the opposite beneficial effect in CsA-treated rats. VEGF mRNA and protein expressions increased with CsA, further increased with L-NAME and became significantly reduced with L-Arg. Flt-1 expression was similar in all groups. On the other hand, KDR/Flk-1 mRNA expression was modulated in a fashion similar to VEGF. Also, nitric oxide modulation did not have an effect on VH-treated rats. CONCLUSIONS: VEGF expression in chronic CsA nephrotoxicity is increased by nitric oxide blockade and decreased by nitric oxide enhancement. Moreover, VEGF probably exerted its effect via the KDR/Flk-1 receptor. The actions of VEGF in this model remain speculative, but it is probable that VEGF plays a role, either independently or through nitric oxide, in CsA-induced fibrosis.