Localization of Fusobacterium nucleatum in oral squamous cell carcinoma and its possible directly interacting protein molecules: A case series.

INTRODUCTION: While 15 to 20% of cancers are associated with microbial infection, the relationship between oral microorganisms and oral squamous cell carcinoma (OSCC) remains unclear. The location of bacteria in a tumor is closely related to its carcinogenic mechanism. The aim of this study was to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues, locate target bacteria, and search for proteins that may interact with target bacteria. MATERIALS AND METHODS: The 16S rDNA method was used to analyse bacterial diversity in clinical OSCC tissue samples and tumor distant normal tissues. Correlations between Fusobacterium abundance and clinicopathological characteristics were analysed using the χ2 test. The position of target bacteria was analysed by fluorescence in situ hybridization (FISH), and the expression of CK, CD31, CD45, CD68, cyclin D1, ß-catenin, E-cadherin, NF-κB, and HIF-1α was analysed by immunohistochemistry (IHC) in OSCC tumor tissues and tumor distant normal tissues. RESULTS: The 16S rDNA results showed that the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues. High expression of Fusobacterium was significantly correlated with the lifestyle-related oral risk habits, including smoking (p=0.036) and alcohol consumption (p=0.022), but did not correlate with patient sex, age, tumor laterality, tumor size, grade or TNM stage. Fusobacterium nucleatum was enriched in tumor stroma, where CD31+ blood vessels and inflammatory cells (including CD45+ leukocytes and CD68+ macrophages) were densely distributed. Cyclin D1 was mainly expressed in the nucleus of tumor cells. ß-catenin was expressed in the tumor cell membrane and was positively expressed in tumor interstitial vascular endothelial cells. E-cadherin was mainly expressed in tumor cell membranes. NF-κB was positively expressed in the cytoplasm of tumor cells, tumor interstitial cells and myo-fibrocytes. HIF-1α was mainly expressed in the cytoplasm of tumor interstitial cells. HIF-1α was highly expressed where Fusobacterium nucleatum was densely distributed. CONCLUSION: According to our study, the detected amount of Fusobacterium in OSCC tumor tissues was significantly larger than that in tumor distant normal tissues, and Fusobacterium nucleatum might aggravate inflammation and hypoxia by interacting with NF-κB and HIF-1α in OSCC.

Promotion of osteogenesis in BMSC under hypoxia by ATF4 via the PERK-eIF2α signaling pathway.

Mandibular distraction osteogenesis (MDO) is an endogenous tissue engineering technology in which bone marrow mesenchymal stem cells (BMSC) play a key role in MDO-related osteogenesis. Activating transcription factor 4 (ATF4) is involved in osteogenesis through activation of PERK (Protein kinase R-like endoplasmic reticulum kinase) in endoplasmic reticulum stress (ERS) condition under hypoxia. However, the specific role of ATF4 in MDO with BMSC remains unknown. The aim of this study was to explore the effects of ATF4 in MDO with BMSC under hypoxia. Briefly, canine BMSCs were cultured in a hypoxic chamber, and effects of hypoxia were evaluated using cell migration assay and Alizarin Red S staining. Expression levels of protein kinase R-like endoplasmic reticulum kinase, eukaryotic translation initiation factor 2α, ATF4, osteocalcin, and bone sialoprotein were evaluated using quantitative polymerase chain reaction and western blotting. BMSCs were transduced with the ATF4-small interfering RNA lentivirus. The effects were evaluated using all the aforementioned experiments. The results showed that hypoxia promoted migration, osteoblast differentiation, and ATF4 expression in BMSC. ATF4 knockdown in BMSC significantly inhibited migration and osteoblast differentiation abilities, while hypoxia reversed these effects to some extent. In addition, the molecular mechanism partly depended on the ERS signaling pathway, with ATF4 as the key factor. In summary, we presented a novel mechanism of ATF4-mediated regulation of BMSC under hypoxia.

Hsp20 Promotes Endothelial Progenitor Cell Angiogenesis via Activation of PI3K/Akt Signaling Pathway under Hypoxia.

BACKGROUND: Mandibular distraction osteogenesis (MDO) is a kind of endogenous tissue engineering technology that lengthens the jaw and opens airway so that a patient can breathe safely and comfortably on his or her own. Endothelial progenitor cells (EPCs) are crucial for MDO-related angiogenesis. Moreover, emerging evidence suggests that heat shock protein 20 (Hsp20) modulates angiogenesis under hypoxic conditions. However, the specific role of Hsp20 in EPCs, in the context of MDO, is not yet known. The aim of this study was to explore the expression of Hsp20 during MDO and the effects of Hsp20 on EPCs under hypoxia. METHODS: Mandibular distraction osteogenesis and mandibular bone defect (MBD) canine model were established. The expression of CD34, CD133, HIF-1α, and Hsp20 in callus was detected by immunofluorescence on day 14 after surgery. Canine bone marrow EPCs were cultured, with or without optimal cobalt chloride (CoCl2) concentration. Hypoxic effects, caused by CoCl2, were evaluated by means of the cell cycle, cell apoptosis, transwell cell migration, and tube formation assays. The Hsp20/KDR/PI3K/Akt expression levels were evaluated via immunofluorescence, RT-qPCR, and western blot. Next, EPCs were incorporated with either Hsp20-overexpression or Hsp20-siRNA lentivirus. The resulting effects were evaluated as described above. RESULTS: CD34, CD133, HIF-1α, and Hsp20 were displayed more positive in the callus of MDO compared with MBD. In addition, hypoxic conditions, generated by 0.1 mM CoCl2, in canine EPCs, accelerated cell proliferation, migration, tube formation, and Hsp20 expression. Hsp20 overexpression in EPCs significantly stimulated cell proliferation, migration, and tube formation, whereas Hsp20 inhibition produced the opposite effect. Additionally, the molecular mechanism was partly dependent on the KDR/PI3K/Akt pathway. CONCLUSION: In summary, herein, we present a novel mechanism of Hsp20-mediated regulation of canine EPCs via Akt activation in a hypoxic microenvironment.

Hypoxia improved vasculogenesis in distraction osteogenesis through Mesenchymal-Epithelial transition (MET), Wnt/ß-catenin signaling pathway, and autophagy.

INTRODUCTION: The osteogenesis rate of distraction osteogenesis is 4-6 times faster than that of infants, far beyond fracture healing. However, the osteogenesis mechanism of DO is complicated and inconclusive owing to two significant elements: mechanical tension which is well explored and trauma caused by bone fracture. Vasculogenesis and EPCs are critical for successful bone regeneration during DO. Thus, this study aimed to explore the effects of hypoxia caused by trauma or CoCl2 on the vasculogenesis of DO and EPCs. MATERIAL AND METHODS: Mandibular DO and BF models were generated using 6 beagle dogs with a distraction rate of 1 mm per day for 7 days or acute lengthening for 7 mm. The vasculogenesis in DO-gap or BF-gap were assessed via histological analyses, qRT-PCR and immunofluorescence staining. Dog bone marrow EPCs were isolated and cultured with or without 0.1 mM CoCl2. The effect of hypoxia caused by CoCl2 were subsequently valuated via in vitro assays including Cell Counting Kit-8, transwell assay, qRT-PCR, western blot, and immunofluorescence staining. RESULTS: Histological analyses, qRT-PCR and immunofluorescence staining revealed that vasculogenesis markedly accelerated in DO-gap compared with BF-gap, and the DO-gap displayed more positive to CD133, CD34, HIF-1α, E-cadherin, beclin1, ß-catenin, VEGF, bFGF, and less positive to ZEB1 than BF-gap. In addition, in vitro analyses revealed CoCl2 treatment enhanced EPCs proliferation and migration, and the levels of HIF-1α, E-cadherin, ß-catenin, beclin1, VEGF, bFGF of EPCs were increased, but the level of ZEB1 was decreased. CONCLUSION: Our studies showed that hypoxia promoted vasculogenesis in DO and EPCs, and the mechanism may involve autophagy, Wnt/ß-catenin signaling pathway, and Mesenchymal-Epithelial transition (MET).

Prognostic Value of Gai's Plaque Score and Agatston Coronary Artery Calcium Score for Functionally Significant Coronary Artery Stenosis.

BACKGROUND: The prognostic values of the coronary computed tomography angiography (CCTA) score for predicting future cardiovascular events have been previously demonstrated in numerous studies. However, few studies have used the rich information available from CCTA to detect functionally significant coronary lesions. We sought to compare the prognostic values of Gai's plaque score and the coronary artery calcium score (CACS) of CCTA for predicting functionally significant coronary lesions, using fractional flow reserve (FFR) as the gold standard. METHODS: We retrospectively analyzed 107 visually assessed significant coronary lesions in 88 patients (mean age, 59.6 ± 10.2 years; 76.14% of males) who underwent CCTA, invasive coronary angiography, and invasive FFR measurement. An FFR <0.80 indicated hemodynamically significant coronary stenosis. Lesions were divided into two groups using an FFR cutoff value of 0.80. We compared Gai's plaque scores and CACS between the two groups and evaluated the correlations of these scores with FFR. The statistical methods included unpaired t-test, Mann-Whitney U-test, and Spearman's correlation coefficients. RESULTS: Coronary lesions with FFR <0.80 had higher Gai's scores than those with FFR ≥0.80. Gai's score had the strongest correlation with FFR (r = -0.48, P < 0.01) and had a greater area under the curve = 0.72 (95% confidence interval: 0.61-0.82; P < 0.01) than the CACS of whole arteries and a single artery. CONCLUSIONS: Both CACS in a single artery and Gai's plaque score demonstrated a good capacity to assess functionally significant coronary artery stenosis when compared to the gold standard FFR. However, Gai's plaque score was more predictive of FFR <0.80. Gai's score can be easily calculated in daily clinical practice and could be used when considering revascularization.

Long-term MMP inhibition by doxycycline exerts divergent effect on ventricular extracellular matrix deposition and systolic performance in stroke-prone spontaneously hypertensive rats.

Pharmacologic inhibition of matrix metalloproteinases (MMP) by doxycycline is of therapeutic potential for a number of cardiovascular diseases characterized by excessive activation of MMP. So far, long-term administration of doxycycline in the treatment of hypertensive ventricular remodeling has not been systemically investigated. Seven-week-old stroke-prone spontaneously hypertensive rats (SHRSP) were fed with doxycycline (30 mgKg(-1) daily) for 26 weeks, when the mortality rate of the control group reached 50%. Stroke incidence was recognized by daily monitoring of stroke symptoms. Left ventricular (LV) performance was measured by in-vivo pressure-volume loop analysis and ex-vivo passive pressure-volume relationship at the time of sacrifice. Collagen deposition, gelatinases activity, protein abundance of gelatinases, and tissue inhibitor of matrix metalloproteinases (TIMP) -1, -2, and related mRNA levels in the heart were determined. MMP-9 expression was not detected in all groups. Excessive activation of MMP-2 in the heart could be partially suppressed by doxycycline. Left ventricular systolic function and ventricular size was partially ameliorated by doxycycline; however, elevated collagen deposition co-existed in the heart. Moreover, doxycycline could downregulate MMP-2 and TIMP-1 expression both at mRNA and protein levels, and TIMP-2 represented opposite expression pattern. These results demonstrate that long-term administration of doxycycline during the development of hypertension has no impact on stroke death, and could partially preserve LV systolic performance and restrain LV chamber dilation, but leads to increased LV extracellular matrix accumulation. Interrupted cardiac MMP-2/TIMP-2 balance by doxycycline may play a role in this process.

High salt intake accelerated cardiac remodeling in spontaneously hypertensive rats: time window of left ventricular functional transition and its relation to salt-loading doses.

Salt-loading is an accelerator of hypertensive left ventricular (LV) remodeling. The relationship between salt-loading doses and the time window in which a transition from compensated to decompensated LV hypertrophy occurs in spontaneously hypertensive rats (SHR) is unclear. Eight-week-old male SHR and Wistar Kyoto rats (WKY) were randomized to receive normal (0.5% NaCl) and high salt diets (4% or 8% NaCl) for 12 weeks. Left ventricular remodeling was dynamically determined by echocardiography. LV invasive hemodynamics and morphologic staining [collagen deposition, cardiomyocte hypertrophy, DNA damage (8-hydroxy-2-deoxyguanosine, 8-OHdG) and apoptosis] were performed at time of sacrifice. Cardiac malonyldialdehyde (MDA) level was measured by ELISA. No differences between 4% and 8% salt diets, in terms of blood pressure (BP) levels, heart mass index, and myocardial fibrosis were observed either in SHR or in WKY. In high salt-loaded SHR, the LV ejection fraction and wall thickness peaked at 8 weeks after salt-loading, parallel with a progressive enlargement of the LV chamber size. Furthermore, when compared to 4% salt SHR, LV functions were significantly compromised in 8% salt SHR, accompanied by more prominent cardiomyoctye hypertrophy, oxidative stress (and related DNA damage), and apoptosis. Salt-loading for 12 weeks with 8% NaCl diet is more efficient to induce LV dysfunction than 4% NaCl diet does in SHR, possibly by initiating increased oxidative stress and resultant cardiac damage. Moreover, 8 to 12 weeks after 8% salt-loading is the key time window in which a transition from compensated to decompensated LV hypertrophy occurs.

Postinfarction healing dynamics in the mechanically unloaded rat left ventricle.

The healing process is a key determinant for postinfarction left ventricular (LV) remodeling and the development of heart failure, which could be influenced by mechanical (pressure and/or volume) load. So far, limited information exists regarding an indepth characterization of the postinfarct healing process in the mechanically unloaded state. In the present work, we performed isogenic Lewis-to-Lewis rat abdominal heterotopic heart transplantation, which is characterized by hemodynamic unloading in the left ventricle, and simultaneously ligated the left anterior descending coronary artery (T-infarct group). Pathological evolution was dynamically compared with that of in situ infarcted Lewis hearts (I-infarct group) on days 3, 7, 14, and 35. There was a remarkable myocardial salvage in the unloaded heart, as shown by the improvement in infarct size (T-infarct group: 25.47% ± 4.31% vs. I-infarct group: 38.46% ± 4.82%, P < 0.01) and the smaller fraction of fibrosis in infarct segments (T-infarct group: 42.12% ± 8.40% vs. I-infarct group: 75.65% ± 10.51%, P < 0.01). In addition, there was a progressive disorganization of the two-dimensional collagen fiber alignment as well as retarded collagen fiber maturation in the T-infarct group. We also observed enhanced angiogenesis, lymphangiogenesis, and inflammatory cell retention in the infarct region during mechanical unloading. Moreover, capillary density and collagen deposition were significantly increased in the noninfarcted area of the unloaded heart compared with the same region in the in situ infarcted heart. In conclusion, ischemic insult in the mechanically unloaded heart elicits an altered inflammatory and healing response, which is characterized by myocardial salvage, delayed resolution of inflammation, and disorganization of the collagen orientation in the infarcted region. These findings could provide novel insights into the contribution of hemodynamic load in the postinfarction healing process. Further studies are warranted to elucidate its potential mechanism.