Skin wound healing in the SKH-1 female mouse following inducible nitric oxide synthase inhibition

BACKGROUND: The inducible isoform of the nitric oxide (NO) synthase (NOS) enzyme (iNOS) is upregulated by inflammatory mediators and/or other pathological stresses, generating high, sustained levels of NO. Cumulative data suggest a role for NO in the regulation of skin wound healing, although it is not clear to what extent NO generated by iNOS, and possibly endothelial NOS (eNOS), contribute to that healing process. Because of the current lack of understanding regarding the contribution of iNOS in wound healing, as well as the lack of wound healing data available for SC-842, an iNOS inhibitor, this in vivo study was conducted to investigate the possible role of SC-842 in interfering with wound healing. OBJECTIVES: This study evaluated whether inhibition of iNOS affects incisional skin wound healing. METHODS: Using a cutaneous full-thickness, sutured, incisional wound model in hairless SKH-1 mice, the role of iNOS in the wound healing process was evaluated by comparing in vivo effects of the iNOS inhibitor, SC-842, at various doses that result in selective inhibition of iNOS as well as nonselective NOS inhibition (as evidenced by elevated blood pressure resulting in inhibition of eNOS and/or neuronal NOS). Dexamethasone was used as a positive control. RESULTS: There were no differences in wound healing at day 28 postwounding, as evaluated by tensile strength and histology, between SC-842- and vehicle-treated animals. A decrease in tensile strength was noted at day 14 postwounding in wounds from the mid- and high-dose-treated animals as compared with vehicle-treated animals, but this difference was slight and was not associated with histological differences from vehicle-treated controls. CONCLUSIONS: These data indicate that iNOS inhibition does not adversely affect the healing of incisional wounds in SKH-1 mice as assessed over 28 days by wound tensile strength and histology.     

Human keratinocytes release high levels of inducible heat shock protein 70 that enhances peptide uptake

Abstract: Background: The stress‐inducible chaperone heat shock protein (HSP) 70 is considered a ‘danger signal’ if released into the extracellular environment. It has been proposed to play a role in the pathogenesis of skin diseases such as psoriasis and lupus erythematosus (LE). Objectives: The aim of this study was to decipher the role of human primary keratinocytes with regard to release and reactivity to HSP70. Methods: We determined HSP70 and IFNγ in cell supernatants by ELISA. Uptake of labelled HSP70 or labelled peptide by human primary keratinocytes or macrophages was analysed by flow cytometry and fluorescent microscopy. Results: We found that living keratinocytes are an important source of HSP70 in the skin compartment. They release considerably more HSP70 than fibroblasts, macrophages or lymphocytes. Interestingly, keratinocytes also bind and internalise HSP70/HSP70–peptide complexes. TNFα, IL‐27 as well as HMGB‐1 enhanced the uptake of HSP70. No difference with regard to HSP70 release or uptake was observable between keratinocytes from healthy donors or patients with cutaneous LE. Keratinocytes pulsed with HSP70–peptide complexes significantly increased IFNγ production by autologous T cells. Conclusions: Production and uptake of inducible HSP70 by keratinocytes may critically influence the chronic course of inflammatory skin diseases.     

Lipopolysaccharide and cytokines induce nitric oxide synthase and produce nitric oxide in cultured normal human melanocytes

Abstract The role of nitric oxide in normal and pathological conditions of human skin is still poorly understood. In this study we have demonstrated by immunobloting the expression of an inducible nitric oxide synthase isoform (iNOS) in cultured normal human melanocytes treated with bacterial lipopolysaccharide, tumor necrosis factor-· and interferon-á. Nitric oxide was also detected in the culture medium and its formation was abolished upon treatment with N^G-monomethyl-l-arginine(l-NMMA), a competitive inhibitor of nitric oxide synthase. These results suggest that nitric oxide could led to autodestruction of melanocytes causing skin depigmentation. The therapeutic relevance of nitric oxide synthase inhibitors in treatment of vitiligo was suggested. Received: 15 September 2000 / Revised: 7 October 2000 / Accepted: 20 January 2001     

Extremely low frequency electromagnetic fields modulate expression of inducible nitric oxide synthase,endothelial nitric oxide synthase and cyclooxygenase‐2 in the human keratinocyte cell line HaCat: potential therapeutic effects in wound healing

Background Extremely low frequency (ELF) electromagnetic fields (EMF) are known to produce a variety of biological effects. Clinical studies are ongoing using EMF in healing of bone fractures and skin wounds. However, little is known about the mechanisms of action of ELF‐EMF. Several studies have demonstrated that expression and regulation of nitric oxide synthase (NOS) and cyclooxygenase‐2 (COX‐2) are vital for wound healing; however, no reports have demonstrated a direct action of ELF‐EMF in the modulation of these inflammatory molecules in human keratinocytes. Objectives The present study analysed the effect of ELF‐EMF on the human keratinocyte cell line HaCaT in order to assess the mechanisms of action of ELF‐EMF and to provide further support for their therapeutic use in wound healing. Methods Exposed HaCaT cells were compared with unexposed control cells. At different exposure times, expression of inducible NOS (iNOS), endothelial NOS (eNOS) and COX‐2 was evaluated by Western blot analysis. Modulation of iNOS and eNOS was monitored by evaluation of NOS activities, production of nitric oxide (NO) and O₂^? and expression of activator protein 1 (AP‐1). In addition, catalase activity and prostaglandin (PG) E₂ production were determined. Effects of ELF‐EMF on cell growth and viability were monitored. Results The exposure of HaCaT cells to ELF‐EMF increased iNOS and eNOS expression levels. These ELF‐EMF‐dependent increased expression levels were paralled by increased NOS activities, and increased NO production. In addition, higher levels of AP‐1 expression as well as a higher cell proliferation rate were associated with ELF‐EMF exposure. In contrast, ELF‐EMF decreased COX‐2 expression, PGE₂ production, catalase activity and O₂^? production. Conclusions Mediators of inflammation, such as reactive nitrogen and PGE₂, and keratinocyte proliferation are critical for the tissue regenerative processes. The ability of ELF‐EMF to upmodulate NOS activities, thus nitrogen intermediates, as well as cell proliferation, and to downregulate COX‐2 expression and the downstream intermediate PGE₂, highlights the potential therapeutic role of ELF‐EMF in wound healing processes.     

Expression of the inducible isoform of nitric oxide synthase in pigment cell lesions of the skin

Nitric oxide (NO) is a small molecule produced during the conversion of L-arginine to L-citrulline by NO synthase (NOS). Several isoforms of NOS exist, of which the Ca2+-independent, inducible NOS (iNOS or NOS2) is most prominently expressed by macrophages. iNOS activity and increased levels of iNOS have been found in various tumours and tumour cell lines but not in normal tissues; however, the precise role of NO in tumour progression has yet to be elucidated. We studied the expression of iNOS in paraffin sections of 41 benign naevi and 52 primary malignant melanomas (MM) of the skin, as well as in 13 metastatic MM. In addition, nitrotyrosine, indicative of NO production and formation of peroxynitrite, was studied in frozen sections of 13 naevi and 30 MM. Virtually all naevi expressed iNOS, but very few expressed nitrotyrosine, indicating either that iNOS in naevi is functionally inactive, or that naevus cells lack reactive oxygen radicals and thus do not form peroxynitrite. Normal melanocytes in adjacent uninvolved skin were unreactive for both markers. In MM, iNOS was most frequently expressed in the 'pure' and 'invasive' radial growth phase (RGP), whereas expression in the vertical growth phase (VGP) and metastatic phase occurred only in 76% of cases; moreover, in these latest phases of tumour progression, iNOS staining was weak and focal. We conclude that iNOS is expressed de novo in most benign pigment cell lesions. In MM (iNOS-generated) NO appears to play an important part in the early steps of invasion (i.e. the 'invasive' RGP), where it may stimulate neo-angiogenesis and may be a prerequisite for further tumour progression; this view is also supported by the finding of iNOS in the associated blood vessels in the papillary dermis. Finally, our data suggest that (iNOS-generated) NO plays a less significant part in the VGP and in metastatic melanoma.     

Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin

Kang-Rotondo CH, Major S, Chiang TM, Myers LK, Kang ES. Upregulation of nitric oxide synthase in cultured human keratinocytes after ultraviolet B and bradykinin. Photodermatol Photoimmunol Photomed 1996: 12: 57–65. © Munksgaard, 1996. Ultraviolet B (UVB) irradiation of the skin has been reported to upregulate nitric oxide synthase (NOS) activity with enhancement of nitric oxide (NO) formation. Bradykinin, a known stimulator of NO production, is produced in the skin within minutes of UVB irradiation. The combined effect of UVB and bradykinin on NOS was therefore examined in a cultured human keratinocyte (KC) line. Activity was determined in KC homogenates by the recovery of [³H]l -citrulline using labeled l -arginine as the substrate in the presence of mM NADPH. Monoclonal antibodies to specific isoforms of NOS that cross-react with their human counterparts were used to determine the isoform(s) in control, UVB, bradykin treated and UVB and bradykinin treated KC. Human KC express NOS activity which is lowest at confluence and highest during proliferation. UVB increased NOS activity when a set dose of irradiation was administered from 32.2–48.3 mJ/cm² but was inhibitory after 64.4 and 80.5 mJ/cm². Thirty min after 10^?6 M bradykinin, NOS activity nearly doubled followed by return of activity to control levels at 60 min. Activity after UVB and bradykinin was only slightly higher than that observed with bradykinin alone. Immunochemically, an isoform of M_r 155 kDa was detected in control cells with the antibody for the constitutive brain enzyme, bNOS. Recovery of this isoform increased after UVB treatment as well as after bradykinin which was time dependent. When both stimulants were used, the recovery of the 155 kDa enzyme was markedly enhanced, unlike the enzyme activity findings. These data indicate that the expression of NOS activity under unstimulated conditions in human KC in culture is due to the constitutive NOS found in neuronal tissue, bNOS. The recovery of bNOS increased after UVB and after bradykinin while the combination of both resulted in the synergistic increase in bNOS protein with only a marginal further increase in NOS activity.     

诱生型一氧化氮合酶和血管内皮生长因子在皮肤血管瘤中的表达及其与血管增生的关系

目的：研究诱生型一氧化氮合酶（iNOS）及血管内皮生长因子（VEGF）在增生期皮肤血管瘤组织中的表达及相关性。探讨它们与增生期血管瘤血管生成的关系,研究iNOS产生的一氧化氮（NO）和VEGF的相互作用及NO在介导VEGF促血管瘤间质内血管生成中的作用机制。方法：应用免疫组化法检测51例增生期皮肤血管瘤标本中iNOS、VEGF和第Ⅷ因子相关抗原（FⅧRAg）,血管内皮细胞特异性染色计数肿瘤微血管密度（MVD）。结果：①42例血管瘤组织表达VEGF,32例表达iNOS,血管畸形表达较弱或不表达iNOS和VEGF;②VEGF与iNOS的表达呈正相关性：③VEGF、iNOS的表达与血管瘤组织MVD呈正相关性,血管瘤MVD明显高于血管畸形。结论：①VEGF表达与iNOS表达具有明显的相关性,提示iNOS对VEGF的表达和调节血管生成过程中可能具有重要作用;②MVD随着VEGF和iNOS表达的增强而增加,说明两者对血管瘤血管生成具有促进作用。     

The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes

Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.     

一氧化氮合酶在外阴慢性单纯性苔藓和外阴硬化性苔藓中的表达

目的：检测诱导型和内皮型一氧化氮合酶（iNOS 和eNOS）在外阴慢性单纯性苔藓和外阴硬化性苔藓中的表达。方法：采用免疫组织化学SABC法检测iNOS和eNOS在30例外阴慢性单纯性苔藓蜡块（LSC）、30例外阴硬化性苔藓蜡块（LS）和10例外阴正常皮肤中的表达,并以人原始造血细胞抗原（CD34）标记微血管内皮细胞,测量各组织的微血管密度（MVD）。结果：iNOS和eNOS在外阴正常皮肤中无表达;在LSC中iNOS和eNOS每视野平均阳性细胞数分别为14.83±3.79和17.86±4.82,高于LS的8.00±3.35和6.43±3.87,差异均有统计学意义（P＜0.05）;在LSC和正常皮肤中MVD分别为21.58±2.48和20.44±3.66,高于LS（10.34±2.83）。iNOS和eNOS的表达具有明显的正相关性（Kappa=0.811,P＜0.05）。结论：iNOS和eNOS可能与LSC炎症过程中的血管扩张有关;在LS皮损真皮中微血管减少,iNOS和eNOS可代偿性地改善LS的血液循环。     

iNOS在皮肤肿瘤中的表达及其意义

目的:检测诱导型一氧化氮合酶(iNOS)在皮肤良恶性肿瘤中的表达。方法:采用免疫组织化学技术检测25例脂溢性角化病、25例光线性角化病、25例基底细胞癌、30例鳞状细胞癌(I级13例,II-III级17例)、10例正常皮肤组织中iNOS的表达。结果:2例(8.00%)脂溢性角化病、13例(52.00%)光线性角化病、11例(44.00%)基底细胞癌、22例(73.33%)鳞状细胞癌中iNOS呈阳性表达,正常皮肤表达均为阴性。iNOS在鳞状细胞癌、基底细胞癌及光线性角化病中的阳性率明显高于脂溢性角化病组(P<0.01),鳞状细胞癌组与光线性角化病组间无明显差别(P>0.05),与其他各组间差异显著(P<0.01或P<0.05),II III级鳞状细胞癌iNOS表达明显高于I级鳞状细胞癌(P<0.05)。结论:皮肤肿瘤中存在iNOS的表达,其合成的一氧化氮可能在皮肤的癌前病变及恶性肿瘤的发生、发展中起到一定的作用。其表达可能有助于皮肤肿瘤恶性度及预后的判断。     

皮肤恶性黑素瘤中诱导型一氧化氮合酶mRNA及其蛋白的表达

目的探讨皮肤恶性黑素瘤中诱导型一氧化氮合酶（iNOS）的表达及其临床意义。方法免疫组化方法检测30例皮肤恶性黑素瘤患者肿瘤组织中iNOS蛋白的表达，逆转录聚合酶链反应（RT-PCR）检测其中6例患者肿瘤组织和肿瘤邻近正常组织中iNOSmRNA的表达。结果iNOS在肿瘤组织中无论蛋白或mRNA阳性表达率皆明显高于邻近正常组织（P〈0．05）。其中iNOS蛋白在无转移的恶性黑素瘤中阳性表达率为69．2％，有转移的恶性黑素瘤中阳性表达率为100％。结论iNOS表达异常在恶性黑素瘤发生、发展与转移过程中可能起重要作用。     

长波紫外线照射对HaCaT细胞iNOS/NO表达的影响

目的 探讨不同剂量长波紫外线 （UVA） 照射HaCaT细胞后不同时间点诱导型一氧化氮合成酶（iNOS）的表达情况。方法 1 J/cm2、5 J/cm2和10 J/cm2 UVA照射HaCaT细胞后继续培养24 h、48 h和72 h,倒置相差显微镜下观察细胞形态的变化;分别用RT-PCR、Western印迹和Griess法检测HaCaT细胞iNOS mRNA、蛋白及NO的表达。结果 所有UVA剂量组HaCaT细胞iNOSmRNA在光照后24 h有表达,48 h达高峰,72 h后下降,各时间点间表达量差异有统计学意义（P < 0.05）;1 J/cm2 UVA照射后3个时间点均未见iNOS蛋白表达,而5 J/cm2和10 J/cm2 UVA照射后iNOS蛋白在24 h增加,48 h达高峰且显著高于24 h（P < 0.05）,照射后72 h无iNOS蛋白表达。所有UVA剂量组HaCaT细胞NO表达量在24 h升高,48 h显著升高,72 h平稳升高,3个时间点NO表达量均比正常对照组明显增加（P < 0.05）。对照组HaCaT细胞无iNOS mRNA和蛋白表达,NO表达量低。结论 HaCaT细胞iNOS和NO的表达变化与UVA照射存在时间和剂量关系。     

Neural nitric oxide synthase participates in pemphigus vulgaris acantholysis through upregulation of Rous sarcoma,mammalian target of rapamycin and focal adhesion kinase

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease characterized by suprabasal acantholysis produced as a consequence of desmoglein (Dsg) and non‐Dsg autoantibodies binding to several targeting molecules localized on the membrane of keratinocytes. Nitric oxide (NO) may exert a pathogenic function in several immunological processes. We have previously demonstrated that neural nitric oxide synthase (nNOS) plays part in PV acantholysis. Also, our group has described a relevant role for HER [human epidermal growth factor receptor (EGFR) related] isoforms and several kinases such as Src (Rous sarcoma), mammalian target of rapamycin (mTOR) and focal adhesion kinase (FAK), as well as caspases in PV development. Using a passive transfer mouse model of PV, we aimed to investigate the relationship between the increase in nNOS and EGFR, Src, mTOR and FAK kinase upregulation observed in PV lesions. Our results revealed a new function for nNOS, which contributes to EGFR‐mediated PV acantholysis through the upregulation of Src, mTOR and FAK. In addition, we found that nNOS participates actively in PV at least in part by increasing caspase‐9 and caspase‐3 activities. These findings underline the important issue that in PV acantholysis, caspase activation is a nNOS‐linked process downstream of Src, mTOR and FAK kinase upregulation.     

Attenuation of contact hypersensitivity by cell‐permeable heat shock protein 70 in BALB/c mouse model

 In contact hypersensitivity (CHS), multiple cells, inflammatory mediators and cytokines are known to be involved in the regulation of the immune response. Previously, we revealed the reactive oxygen species generation by 2, 4, 6‐trinitrobenzene sulphonic acid (TNBS) in vivo, followed by heat shock protein 70 (Hsp70) carbonylation and the exogenous antioxidant role of cell‐permeable Hsp70. Here, we demonstrate the role of Hsp70 using cell‐permeable Hsp70 in the mouse CHS model. Pretreatment of cell‐permeable Hsp70: (i) suppressed ear swelling; (ii) down‐regulated phosphorylated p38, but up‐regulated phosphorylated extracellular signal‐regulated kinase; (iii) increased population of CD4⁺CD25⁺Foxp3⁺ T cells; (iv) decreased secretion of tumor necrosis factor‐α (TNF‐α), IL‐12, interferon‐γ and IL‐2 and (v) but up‐regulated IL‐4 and transforming growth factor beta (TGF‐β) in the lymph nodes. In conclusion, cell‐permeable Hsp70 attenuates CHS through modulation of MAPK pathway and regulation of Th1, Th2 and regulatory T cells.     

Nitric oxide enhances expression of raf kinase inhibitor protein in keratinocytes

Abstract:  Several reports have focused on the potential of nitric oxide (NO) to influence the proliferation and differentiation cascade in a number of mammalian cells. The purpose of this study was to determine the relationship between expression of raf kinase inhibitor protein (RKIP) and proliferation in keratinocyte with NO treatment. Normal human keratinocytes were treated with SNAP (NO donor) doses of 10⁻⁷, 10⁻⁶, 10⁻⁵, 10⁻⁴ and 0  m (control group) separately. Expression of protein and mRNA of RKIP, cell proliferation and apoptosis have been measured. These results showed that elevated expression of RKIP in keratinocyte with NO treatment may contribute to the pathological and physiological features of NO-inhibited proliferation.     

Differential expression of nitric oxide synthases in human scalp epidermal and hair follicle pigmentary units: implications for regulation of melanogenesis

BACKGROUND: Nitric oxide (NO) is a ubiquitous gaseous lipophilic molecule generated from the conversion of L-arginine to L-citrulline by the NO synthases (NOSs). Ultraviolet radiation (UVR)-induced NO production appears to stimulate epidermal melanogenesis. However, given their relative protection from UVR, it is unclear whether NO plays a similar role in hair bulb melanocytes. OBJECTIVES: We aimed to identify the expression profiles of the NOS isoforms endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) and of phosphorylated eNOS and nitrotyrosine within the epidermal and follicular melanin units of normal human haired scalp during the hair growth cycle. METHODS: This study employed single and double immunohistochemical and immunofluorescence staining techniques using haired scalp from 10 healthy individuals (six women and four men). RESULTS: Melanocytes in the basal layer of the epidermis expressed eNOS, nNOS and nitrotyrosine. By contrast, melanogenically active melanocytes of the anagen hair bulb were wholly negative for these markers. However, other follicular melanocytes not actively involved in pigment production, including undifferentiated melanocytes located in the outer root sheath and melanocytes surviving the apoptosis-driven hair follicle (HF) regression during catagen/telogen, expressed eNOS, nNOS and nitrotyrosine. While iNOS was only weakly expressed in the basal layer of the human epidermis, it was highly expressed in keratinocytes of the inner root sheath (IRS), where it colocalized with trichohyalin, a differentiation-associated protein of the IRS that requires enzyme-catalysed conversion of arginine to citrulline. CONCLUSIONS: The NOS isoforms and nitrotyrosine are differentially expressed in different cutaneous melanocyte subpopulations. Results of this study suggest a possible role for eNOS, nNOS, iNOS and nitrotyrosine in melanocyte biology, particularly with respect to melanogenesis and melanocyte survival during HF regression. Another example of possible NO involvement in HF biology is the postsynthetic modification of trichohyalin in differentiating keratinocytes of the IRS. These results suggest that NO may influence several aspects of HF biology.