Assessment of Unicuspid Aortic Valve Stenosis Using Multimodality Imaging, X-ray Radiography and Raman Analysis.

Unicuspid aortic valve (UAV) is an extremely rare form of congenital cardiac malformation, leading to aortic stenosis (AS), aortic regurgitation (AR), or both. We report the case of a 55-year-old man with unicommissural UAV associated with severe AS and mild AR using different multimodality imaging approaches. The excised UAV isolated after aortic valve replacement exhibited an eccentric "teardrop" opening with a slit-shaped unicommissural structure. Raman spectroscopic results indicated that 3 unevenly distributed components were deposited on the surface of the UAV, in which calcium hydroxyapatite and type-B carbonate apatite were the predominate components deposited on the surface, leading to severe AS formation.

Echocardiographic manifestations and chemical composition of stenotic bicuspid aortic valves.

Bicuspid aortic valve (BAV) is an inherited form of heart disease with only two aortic valve leaflets via a disorder of cardiac valvulogenesis. We investigated the in vivo echocardiographic features of cardiac morphology in patients with BAV and the ex vivo compositional components of all the excised BAV leaflets isolated from BAV patients. Three BAV patients were randomly selected. All patients underwent 2D transthoracic echocardiography (TTE) with a Doppler ultrasound tool. The compositional components of each respective BAV leaflet for all the excised BAVs were determined by a portable fiber-optic Raman spectroscopy. Preoperative TTE revealed the thickened and calcified BAV leaflets, and stenotic aortic flow for all BAV patients. These BAV patients exhibited severe aortic stenosis (AS) by the lower values of aortic valve area (AVA) index. One patient showed a more significant left ventricle hypertrophy, whereas two patients exhibited a significant aortic regurgitation (AR). In addition, three different Raman spectral patterns were summed up from 121 randomized Raman determinations for all the excised BAV leaflets. The main calcified deposition in each BAV leaflet was formed by large amounts of calcium hydroxyapatite and type-B carbonate apatite (Raman bands at 960 and 1070 cm-1). The calcified BAV leaflets were composed of different compositional components such as calcium hydroxyapatite, type-B carbonate apatite, lipids, proteins, cholesterol and ß-carotene. The rare NL subtype of type 1 BAV morphotype was found in one patient, but two patients had the purely BAV morphotype with two equal-sized leaflets.

Cost-utility analysis of direct ventricular assist device vs double bridges to heart transplantation in patients with refractory heart failure.

OBJECT: This study compared the cost-utility of direct ventricular assist device (VAD) vs double bridges, extracorporeal membrane oxygenation (ECMO) before VAD, to heart transplantation in patients with refractory heart failure. MATERIALS AND METHODS: From a health payer perspective, a Markov model was developed. The cycle length was 1 month, and the time horizon was a lifetime. Probabilities and direct cost data were calculated from a nationwide claim database. Utility inputs were adopted from published sources. The utility was expressed as quality-adjusted life years (QALYs). Both costs and utility were discounted by an annual rate of 3%. Deterministic and probabilistic sensitivity analyses were performed to test the stability of the model. RESULTS: The direct VAD group had less lifetime costs (USD 95 910 vs USD 129 516) but higher lifetime QALYs than the double bridges group (1.73 vs 0.89). The sensitivity analysis revealed that the direct VAD group consistently had lower cost and higher QALYs during all variations in model parameters. The probability that direct VAD was cost-effective exceeded 75% at any levels of willing-to-pay. CONCLUSION: From a health insurance payer perspective, direct VAD bridge to heart transplantation appeared to be more cost-effective than double bridges in patients with refractory heart failure.

Dual-Energy Computed Tomography Gemstone Spectral Imaging: A Novel Technique to Determine Human Cardiac Calculus Composition.

OBJECTIVE: Understanding the chemical composition of any calculus in different human organs is essential for choosing the best treatment strategy for patients. The purpose of this study was to assess the capability of determining the chemical composition of a human cardiac calculus using gemstone spectral imaging (GSI) mode on a single-source dual-energy computed tomography (DECT) in vitro. METHODS: The cardiac calculus was directly scanned on the Discovery CT750 HD FREEdom Edition using GSI mode, in vitro. A portable fiber-optic Raman spectroscopy was also applied to verify the quantitative accuracy of the DECT measurements. RESULTS: The results of spectral DECT measurements indicate that effective Z values in 3 designated positions located in this calculus were 15.02 to 15.47, which are close to values of 15.74 to 15.86, corresponding to the effective Z values of calcium apatite and hydroxyapatite. The Raman spectral data were also reflected by the predominant Raman peak at 960 cm for hydroxyapatite and the minor peak at 875 cm for calcium apatite. CONCLUSIONS: A potential single-source DECT with GSI mode was first used to examine the morphological characteristics and chemical compositions of a giant human cardiac calculus, in vitro. The CT results were consistent with the Raman spectral data, suggesting that spectral CT imaging techniques could be accurately used to diagnose and characterize the compositional materials in the cardiac calculus.

Application of scanning electron microscopy and X-ray microanalysis: FE-SEM, ESEM-EDS, and EDS mapping for studying the characteristics of topographical microstructure and elemental mapping of human cardiac calcified deposition.

To explore the pathogenic mineral formation in a huge cardiolith isolated from the left heart atrium of an 80-year-old male patient, field emission scanning electron microscopy (FE-SEM) was used to analyze the topographic microstructure and perform elemental mapping in a cross-section of the cardiac calcified deposit after dissection. Environmental SEM equipped with an energy dispersive X-ray spectrometer (EDS) was also used to investigate the composition and spatial distribution of elements in the cross-section, and fiberoptic Raman spectroscopy was used to reidentify the chemical composition of designated positions. The results indicated that calcium hydroxyapatite and cholesterol were the main components of the cardiac calculus. The plate-like structures of calcium hydroxyapatite were unevenly spread over the cholesterol of the cardiac calculus. The calcium hydroxyapatite-rich area exhibited higher amounts of C, O, P, and Ca elements as well as trace amounts of N, Na, Mg, and Al, whereas the major concentration of C, minor concentrations of N and O, and trace amounts of P and Ca were observed in the cholesterol-rich area. Hypercholesterolemia associated with calcification of this cardiac calculus was proposed. Both FE-SEM and ESEM energy dispersive X-ray microanalyses were performed directly, for the first time, to provide useful information on the microstructural characteristics and spatial distribution of elements on the surface of human cardiac calculi.

Dysregulation of mitochondrial dynamics mediated aortic perivascular adipose tissue-associated vascular reactivity impairment under excessive fructose intake.

Excessive fructose intake presents the major risk factor for metabolic cardiovascular disease. Perivascular adipose tissue (PVAT) is a metabolic tissue and possesses a paracrine function in regulating aortic reactivity. However, whether and how PVAT alters vascular function under fructose overconsumption remains largely unknown. In this study, male Sprague-Dawley rats (8 weeks old) were fed a 60% high fructose diet (HFD) for 12 weeks. Fasting blood sugar, insulin, and triglycerides were significantly increased by HFD intake. Plasma adiponectin was significantly enhanced in the HFD group. The expression of uncoupling protein 1 (UCP1) and mitochondrial mass were reduced in the aortic PVAT of the HFD group. Concurrently, the expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and mitochondrial transcription factor A (TFAM) were suppressed. Furthermore, decreased fusion proteins (OPA1, MFN1, and MFN2) were accompanied by increased fission proteins (FIS1 and phospho-DRP1). Notably, the upregulated α-smooth muscle actin (α-SMA) and osteocalcin in the PVAT were concurrent with the impaired reactivity of aortic contraction and relaxation. Coenzyme Q10 (Q, 10 mg/100 mL, 4 weeks) effectively reversed the aforementioned events induced by HFD. Together, these results suggested that the dysregulation of mitochondrial dynamics mediated HFD-triggered PVAT whitening to impair aortic reactivity. Fortunately, coenzyme Q10 treatment reversed HFD-induced PVAT whitening and aortic reactivity.

High fructose induced osteogenic differentiation of human valve interstitial cells via activating PI3K/AKT/mitochondria signaling.

BACKGROUND: Aortic valve stenosis (AS) is a common, lethal cardiovascular disease. There is no cure except the valve replacement at last stage. Therefore, an understanding of the detail mechanism is imperative to prevent and intervene AS. Metabolic syndrome (MetS) is one of the major risk factors of AS whereas fructose overconsuming tops the list of MetS risk factors. However, whether the fructose under physiological level induces AS is currently unknown. METHODS: The human valve interstitial cells (hVICs), a crucial source to develop calcification, were co-incubated with fructose at 2 or 20 mM to mimic the serum fructose at fasting or post-fructose consumption, respectively, for 24 h. The cell proliferation was evaluated by WST-1 assays. The expressions of osteogenic and fibrotic proteins, PI3K/AKT signaling, insulin receptor substrate 1 and mitochondrial dynamic proteins were detected by Western blot analyses. The mitochondrial oxidative phosphorylation (OXPHOS) was examined by Seahorse analyzer. RESULTS: hVICs proliferation was significantly suppressed by 20 mM fructose. The expressions of alkaline phosphatase (ALP) and osteocalcin were enhanced concurrent with the upregulated PI3K p85, AKT, phospho(p)S473-AKT, and pS636-insulin receptor substrate 1 (p-IRS-1) by high fructose. Moreover, ATP production capacity and maximal respiratory capacity were enhanced in the high fructose groups. Synchronically, the expressions of mitochondrial fission 1 and optic atrophy type 1 were increased. CONCLUSIONS: These results suggested that high fructose stimulated the osteogenic differentiation of hVICs via the activation of PI3K/AKT/mitochondria signaling at the early stage. These results implied that high fructose at physiological level might have a direct, hazard effect on the progression of AS.

Clinical features of cytomegalovirus anterior uveitis in immunocompetent patients.

PURPOSE: To describe the clinical presentation of cytomegalovirus (CMV) anterior uveitis in human immunodeficiency virus (HIV)-negative patients. DESIGN: Retrospective, interventional case series. METHODS: HIV-negative patients with anterior uveitis associated with elevated intraocular pressure (hypertensive anterior uveitis) seen at the Singapore National Eye Centre had their aqueous analyzed for viral deoxyribonucleic acid by polymerase chain reaction, and their records were reviewed for demographic data, ocular findings, laboratory results, and treatment. RESULTS: Aqueous was obtained from 105 of 106 eligible eyes. Twenty-four eyes demonstrated positive results for CMV (22.8%). Eighteen eyes had Posner-Schlossman syndrome (PSS; 75%) at presentation, five eyesba had Fuchs heterochromic iridocyclitis (FHI; 20.8%), and one eye had a presumed herpetic anterior uveitis. Twelve of the 24 eyes were treated with ganciclovir. Of the 12 who completed treatment, all responded clinically, and their aqueous demonstrated negative results for CMV on repeat testing. However, nine had recurrences within eight months of stopping treatment and required further courses of ganciclovir. The 81 CMV-negative eyes included 30 with PSS, 11 with FHI, 27 with uveitic glaucomas of unknown cause, and 13 with presumed herpetic anterior uveitis. CONCLUSIONS: CMV anterior uveitis is not uncommon in our immunocompetent patients and it may present as a recurrent acute or chronic inflammation, resembling PSS, herpetic anterior uveitis, or FHI.

New morphological classification of congenital quadricuspid aortic valve and its histopathologic features.

We report a 52-year-old male patient who had a quadricuspid aortic valve (QAV) associated with aortic regurgitation (AR) and left ventricular hypertrophy (LVH). A new accessory cusp (ACC) with maximum thickness than other cusps was located between right coronary cusp (RCC) and left coronary cusp (LCC). The histopathological features revealed markedly thickened and distorted cusp architecture with fibrosis and/or myxomatous degeneration in both non-coronary cusp (NCC) and ACC. Two equal sizes for larger cusps (RCC and NCC) and two equal sizes for smaller cusps (LCC and ACC) were obtained. This QAV belonged to type C QAV of Hurwitz's classification, but also suggested as a modified type III of Jagannath's classification or a new type V of Nakamura's classification by locating ACC between RCC and LCC.

Corneal endotheliitis associated with evidence of cytomegalovirus infection.

OBJECTIVE: To describe the clinical presentation of cytomegalovirus (CMV) corneal endotheliitis in human immunodeficiency virus (HIV)-negative patients. DESIGN: Retrospective interventional case series. PARTICIPANTS: Twelve consecutive patients with corneal endotheliitis diagnosed between 2002 and 2005. METHODS: Aqueous of eyes with corneal endotheliitis was analyzed for viral DNA by polymerase chain reaction (PCR), and patient records were reviewed for demographic data, medical and ocular history, best-corrected Snellen visual acuity, intraocular pressure (IOP), anterior and posterior segment findings, laboratory workup, diagnosis, and treatment. MAIN OUTCOME MEASURE: Presence of CMV DNA. RESULTS: Corneal endotheliitis was seen in 12 eyes of 10 patients during the study period. There were 8 men and 2 women, and all were Chinese. Their mean age was 49 years (range, 25-61 years). The corneal involvement ranged from small areas of focal endotheliitis to diffuse bullous keratopathy. The keratic precipitates had a variable appearance. There was only mild anterior chamber inflammation with no posterior synechiae. Two thirds of eyes had diffuse iris atrophy. All the eyes had elevated IOP. Eleven of the 12 eyes were positive for CMV DNA. None of the patients were positive for HIV. All patients had received local or systemic immunosuppression, or both, before corneal endotheliitis developed. Ten eyes of 8 patients were treated with systemic antiviral therapy. After treatment, the endotheliitis resolved completely in 7 eyes, and 3 eyes had significant improvement in corneal translucency. The IOP was normal, with no medications in all but 1 eye. Repeat PCR analysis in all the treated eyes was negative for CMV DNA. CONCLUSIONS: Cytomegalovirus infection is an important cause of corneal endotheliitis in our patients, and appropriate antiviral therapy may prevent more ocular damage.

Dengue-associated maculopathy.

OBJECTIVE: To describe the clinical spectrum of fundus manifestations and angiographic and optical coherence tomographic features of dengue-associated maculopathy in a large series. METHODS: We reviewed clinical records of patients diagnosed as having dengue maculopathy at the Singapore National Eye Centre between January 1, 2002, and December 31, 2005. RESULTS: We identified 41 patients with serological evidence of dengue fever who had ocular signs and symptoms not attributable to other diseases within 1 month after onset of symptoms of dengue. Seventy-one eyes had maculopathy. Mean best-corrected visual acuity in the affected eye was 20/40 (range, hand motions to 20/20). Intraretinal hemorrhages were seen in 45% of eyes, usually in association with venous sheathing. Fundus fluorescein angiography demonstrated venular occlusion in 25% or arteriolar and/or venular leakage in 3% and 13%, respectively. Yellow subretinal dots were an unusual finding in 28%. Of these, 50% showed corresponding hypofluorescent spots on indocyanine green angiography. Central or paracentral scotomas were observed in 63%. Twenty-eight patients received steroid treatment. Mean visual acuity showed significant improvement between weeks 2 and 4, with an increasing proportion of eyes achieving a best-corrected visual acuity of 20/40 or better across time. CONCLUSION: Fundus fluorescein and indocyanine green angiography, optical coherence tomography, and visual field testing are useful tools in the diagnosis of dengue maculopathy.

Ex vivo assessment of valve thickness/calcification of patients with calcific aortic stenosis in relation to in vivo clinical outcomes.

Calcific aortic stenosis (AS) plays a critical role in the risk of cardiovascular disease. This preliminary study examined the relationship between the ex vivo valve thickness/calcification and in vivo clinical outcomes of Chinese patients with calcific AS. Six Chinese patients (3 patients with tricuspid aortic valves (TAV)) and 3 patients with. bicuspid aortic valves (BAV) with calcific AS undergoing heart valve replacement were initially chosen for this study. In vivo medical imaging of these calcific AS patients was evaluated using computed tomography and echocardiography. The ex vivo measurements including the actual thickness, calcified area and components of the calcified aortic values excised were performed by a digimatic caliper, X-ray equipment with a cellSens imaging analysis and portable Raman spectroscopy, respectively. Six patients were diagnosed with symptomatic moderate-to-severe AS. The thickness of noncoronary (N) leaflet in the excised TAV was significantly thicker than left-coronary (L) leaflet (p < 0.01), and right-coronary (R) leaflet was also thicker than L (p < 0.05), but no significant difference occurred between N and R (p > 0.05). The extent of calcification in the excised TAV was a statistically significant difference between N and L (p < 0.01) and between R and L (p < 0.01), respectively. However, there was no significant difference between R and L in both thickness and calcification for the excised BAV (p > 0.05). The patients No. 1-3 were found to be TAV with partial commissural fusion. The patient No. 4 was classified as a type 1 NL-BAV morphotype, but both patients 5 and 6 were found to be true BAV (type 0 lateral-BAV). Each calcified valve leaflet was composed of apatites, proteins (collagen and proteoglycan), and a small amount of ß-carotene and cholesterol after Raman spectral determination. The calcified nodules of each valve leaflet were predominately identified to be calcium hydroxyapatite and type-B carbonate apatite. However, octacalcium phosphate was also detected in the protein-rich part of calcified valve leaflets. A positive correlation was observed between thickness and calcification for both excised TAV and BAV after ex vivo examinations. Moreover, a negative relationship was obtained among in vivo AVA index, ex vivo thickness and ex vivo calcification for these calcific AS patients.

Down-regulation of adrenal neuronal nitric oxide synthase mRNAs and proteins after deoxycorticosterone acetate-salt treatment in rats.

The aim of this study was to evaluate the possible changes of adrenal neuronal nitrite oxide synthase (nNOS) messenger RNA (mRNA) and protein of rats after deoxycorticosterone acetate (DOCA)-salt treatment. We determined adrenal nNOS expression in 12 vehicle-treated and 13 DOCA-salt-treated rats by in situ hybridization, immunohistochemistry, and multiplex RT-PCR methods. Adrenal nNOS was also detected by Western blot in five vehicle-treated and five DOCA-salt-treated rats. The results showed that adrenal nNOS mRNA and nNOS immunoreactivities were mainly localized in the medulla and some in the regions of zona glomerulosa. DOCA-salt treatment inactivated nNOS mRNA and peptide expression prominent in the adrenal medulla and slight in the zona glomerulosa. The relative quantities of nNOS mRNA in the adrenals of the DOCA-salt-treated group was 8.8-fold decreased. At the same time, the relative quantities of steroid acute regulatory protein mRNA and phenylethanolamine N-methyltransferase mRNA in the adrenals of the DOCA-salt-treated group were significantly decreased. Western blots showed that total adrenal nNOS were 3.7-fold down-regulated after DOCA-salt treatment. Our results indicated that the down-regulation of adrenal nNOS synthesis might be associated with the inactivation of adrenal function in face of volume expansion.

WOX1 is essential for UVB irradiation-induced apoptosis and down-regulated via translational blockade in UVB-induced cutaneous squamous cell carcinoma in vivo.

PURPOSE: We investigated the role of candidate tumor suppressor and proapoptotic WOX1 (also named WWOX, FOR, or WWOXv1) in UVB-induced apoptosis and formation of cutaneous squamous cell carcinomas (SCC). EXPERIMENTAL DESIGN: Expression of WOX1 and family proteins (WWOX) in human primary cutaneous SCCs was examined by immunohistochemistry, in situ hybridization, and reverse transcription-PCR. UVB irradiation-induced WOX1 activation (Tyr33 phosphorylation and nuclear translocation), apoptosis, and cutaneous SCC formation were examined both in vitro and in vivo. RESULTS: Up-regulation of human WOX1, isoform WOX2, and Tyr33 phosphorylation occurred during normal keratinocyte differentiation before cornification and death. Interestingly, significant reduction of these proteins and Tyr33 phosphorylation was observed in nonmetastatic and metastatic cutaneous SCCs (P < 0.001), but without down-regulation of WWOX mRNA (P > 0.05 versus normal controls), indicating a translational blockade of WWOX mRNA to protein. During acute exposure of hairless mice to UVB, WOX1 was up-regulated and activated in epidermal cells in 24 hours. In parallel with the clinical findings in humans, chronic UVB-treated mice developed cutaneous SCCs in 3 months, with significant reduction of WOX1 and Tyr33 phosphorylation and, again, without down-regulation of WWOX mRNA. Human SCC-25 and HaCaT cells were transfected with small interfering RNA-targeting WOX1 and shown to resist UVB-induced WOX1 expression, activation, and apoptosis. CONCLUSIONS: WOX1 is essential for UVB-induced apoptosis and likely to be involved in the terminal differentiation of normal keratinocytes. During UVB-induced cutaneous SCC, epidermal cells have apparently prevented the apoptotic pressure from overexpressed WOX1 by shutting down the translation machinery for WWOX mRNA.

Spectral and morphological classification of different chronic and acute Taiwanese gallstones via FTIR, SEM and ESEM-EDX microanalyses.

BACKGROUND: Gallstone disease is one of the leading upper gastrointestinal surgical problems in different countries. AIMS: To analyze the chronic gallstones and acute gallbladder sludge retrieved from 36 Taiwanese patients. METHODS: FTIR microspectroscopy was used to classify the types of gallstones, and an ESEM-EDX microanalysis was first applied to determine the microstructural features and elemental compositions of the various gallstones. Bacteria presented on the surface of gallstones were also detected by SEM. RESULTS: Four types of gallstones were obtained from these 36 Taiwanese patients: calcium bilirubinate (CaBR) stones (30.6%), cholesterol stones (19.4%), mixed stones including 6 subtypes (47.2%), and acute gallbladder sludge (2.8%) made of CaBR and protein/insoluble biomaterials. Bacteria imprints and bacterial discharges or bacterial biofilms were also found on the surface of gallstones and acute sludge under a SEM observation. ESEM-EDX results revealed that calcium was found to be the main constituent of all of the types of stones except cholesterol stones, and aluminum was also presented in most of the stones and sludge samples. Chloride was only detected in the acute gallbladder sludge. CONCLUSION: FTIR spectra, morphological features, and elemental compositions of the acute gallbladder sludge were different from those of the chronic gallstones.

Compressive C-shaped lamellar keratoplasty: a surgical alternative for the management of severe astigmatism from peripheral corneal degeneration.

OBJECTIVE: To describe a compressive lamellar surgical technique for treating severe astigmatism in peripheral corneal ectasia. DESIGN: Retrospective, noncomparative, interventional case series. PARTICIPANTS: Four eyes of 3 patients with either pellucid or Terrien's marginal corneal degeneration were included in this series. METHODS: C-shaped lamellar keratoplasty using multiple trephines of different sizes, with deliberate undersizing of the donor graft for a controlled compressive effect, was performed on these patients. MAIN OUTCOME MEASURES: Visual acuity outcome and refraction were measured at different intervals at up to 40 months of follow-up. RESULTS: All eyes achieved Snellen visual acuity of 20/40 or better and stable astigmatism ranging from 0 to -2.75 diopter cylinder within 6 months, with no recurrence of corneal thinning or peripheral corneal vascularization. CONCLUSIONS: Compressive C-shaped lamellar keratoplasty is able to reduce severe corneal astigmatism in peripheral corneal ectasia and can result in good visual and refractive outcomes with early visual rehabilitation.

Composition and distribution of elements and ultrastructural topography of a human cardiac calculus.

Trace elements (TEs) may contribute to the formation of calculi or stones or be involved in the aetiopathogenesis of stone diseases. The compositions and spatial distribution of elements from the inner nucleus to outer crust of the cardiac calculus were investigated by energy-dispersive X-ray fluorescence (EDXRF) spectrometer. The surface topograph, distribution map of elements, elemental and chemical compositions were also determined by environmental scanning electron microscope (ESEM)-energy-dispersive X-ray (EDX) analysis. Twenty-five elements were identifiable from 18 positions on the cardiac calculus by EDXRF spectrometer, in which the highest concentrations of toxic TEs (Ni, Pt, Hg, Sn, Pb, W, Au, Al, Si) and higher levels of essential TEs (Ca, Sr, Cr, P) were detected. A moderate positive Pearson's correlation between TEs concentrations of Mg, Ca or P and location differences from centre to periphery in the cardiac calculus was observed. A positive correlation was also found for Ca/Zn and Ca/Cu, indicating the gradual increase of calcium concentration from inner nucleus to outer crust of cardiac calculus. The drop-like nodules/crystals on the surface of petrous part of cardiac calculus were observed from ESEM analysis. ESEM-EDX analysis determined the calculus to be predominantly composed of calcium hydroxyapatite and cholesterol, as indicated by the petrous surface and drop-like nodules/crystals, respectively. This composition was confirmed using a portable Raman analyser. The spatial distribution analysis indicated a gradual increase in Mg, P and Ca concentrations from the inner nucleus to the outer crust of the cardiac calculus. The major chemical compositions of calcium hydroxyapatite and cholesterol were detected on this cardiac calculus.

Dramatic co-activation of WWOX/WOX1 with CREB and NF-kappaB in delayed loss of small dorsal root ganglion neurons upon sciatic nerve transection in rats.

BACKGROUND: Tumor suppressor WOX1 (also named WWOX or FOR) is known to participate in neuronal apoptosis in vivo. Here, we investigated the functional role of WOX1 and transcription factors in the delayed loss of axotomized neurons in dorsal root ganglia (DRG) in rats. METHODOLOGY/PRINCIPAL FINDINGS: Sciatic nerve transection in rats rapidly induced JNK1 activation and upregulation of mRNA and protein expression of WOX1 in the injured DRG neurons in 30 min. Accumulation of p-WOX1, p-JNK1, p-CREB, p-c-Jun, NF-kappaB and ATF3 in the nuclei of injured neurons took place within hours or the first week of injury. At the second month, dramatic nuclear accumulation of WOX1 with CREB (>65% neurons) and NF-kappaB (40-65%) occurred essentially in small DRG neurons, followed by apoptosis at later months. WOX1 physically interacted with CREB most strongly in the nuclei as determined by FRET analysis. Immunoelectron microscopy revealed the complex formation of p-WOX1 with p-CREB and p-c-Jun in vivo. WOX1 blocked the prosurvival CREB-, CRE-, and AP-1-mediated promoter activation in vitro. In contrast, WOX1 enhanced promoter activation governed by c-Jun, Elk-1 and NF-kappaB. WOX1 directly activated NF-kappaB-regulated promoter via its WW domains. Smad4 and p53 were not involved in the delayed loss of small DRG neurons. CONCLUSIONS/SIGNIFICANCE: Rapid activation of JNK1 and WOX1 during the acute phase of injury is critical in determining neuronal survival or death, as both proteins functionally antagonize. In the chronic phase, concurrent activation of WOX1, CREB, and NF-kappaB occurs in small neurons just prior to apoptosis. Likely in vivo interactions are: 1) WOX1 inhibits the neuroprotective CREB, which leads to eventual neuronal death, and 2) WOX1 enhances NF-kappaB promoter activation (which turns to be proapoptotic). Evidently, WOX1 is the potential target for drug intervention in mitigating symptoms associated with neuronal injury.