Endothelial toll‐like receptor 4 maintains lung integrity via epigenetic suppression of p16INK4a

We previously reported that the canonical innate immune receptor toll‐like receptor 4 (TLR4) is critical in maintaining lung integrity. However, the molecular mechanisms via which TLR4 mediates its effect remained unclear. In the present study, we identified distinct contributions of lung endothelial cells (Ec) and epithelial cells TLR4 to pulmonary homeostasis using genetic‐specific, lung‐ and cell‐targeted in vivo methods. Emphysema was significantly prevented via the reconstituting of human TLR4 expression in the lung Ec of TLR4?/? mice. Lung Ec‐silencing of TLR4 in wild‐type mice induced emphysema, highlighting the specific and distinct role of Ec‐expressed TLR4 in maintaining lung integrity. We also identified a previously unrecognized role of TLR4 in preventing expression of p16^INK4a, a senescence‐associated gene. Lung Ec‐p16^INK4a‐silencing prevented TLR4?/? induced emphysema, revealing a new functional role for p16^INK4ain lungs. TLR4 suppressed endogenous p16^INK4a expression via HDAC2‐mediated deacetylation of histone H4. These findings suggest a novel role for TLR4 in maintaining of lung homeostasis via epigenetic regulation of senescence‐related gene expression.     

Assessment of the association between the frequency of micronucleus and p16INK4a/Ki-67 co-expression in patients with cervical intraepithelial lesions

Human papilloma virus (HPV) infection is the main etiological factor for cervical intraepithelial lesions (CIN). An important characteristic of this process is the loss of genome stability. Therefore, it is imperative to use biomarkers of DNA damage caused by genomic instability to identify high risk individuals. We investigated the frequency of micronuclei (MN) in peripheral blood lymphocytes (PBL) of 20 patients, diagnosed as histologically CIN 1 and 10 healthy controls. We also examined the frequency of other nuclear anomalies including nucleoplasmic bridges (NPBs) and nuclear buds (NBUDs) in PBL of patients with CIN 1 and healthy controls, and evaluated the benefits of p16^INK4a and Ki-67 (p16^INK4a/Ki-67) immunohistochemical double staining for identifying cervical squamous cells that express HPV E6/E7 oncogenes. We analyzed the association between the frequency of MN in PBL and the amount of p16^INK4a/Ki-67 co-expression in CIN 1 patients to establish genomic instability. Among CIN 1 subjects, 15% exhibited diffuse p16^INK4a/Ki-67 co-expression and were considered high positive, 25% of the CIN 1 cases exhibited p16^INK4a/Ki-67 co-expression restricted to the lower part of the epithelium and were considered low positive and the remaining 60% of cases were negative. The frequency of MN, NPBs and NBUDs differed significantly among groups. We found a statistically significant positive correlation between p16^INK4a/Ki-67 co-expression and the frequency of MN, NPBs and NBUDs in PBL. Our findings demonstrate the efficacy of p16^INK4a/Ki-67 double immunostaining for histological samples with CIN 1. MN frequency in PBL might be useful for detecting genomic instability in cases of HPV infection and CIN.     

Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

U.S. Choi and D.Y. Kim Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours Objective: To investigate whether Diff‐Quik stained fine needle aspirate smears can be used to evaluate Ki‐67 expression by immunocytochemistry. Methods: Both cytological and histological samples were obtained from 24 dogs with spontaneously developed mammary gland tumours. The cytological and histological specimens were examined by Diff‐Quik and H&E stains, respectively. After examination, both samples were immunostained using the same Ki‐67 antibody. The % Ki‐67 values were calculated based on the percentage of positively stained tumour cells per 500 and 1000 tumour cells in cytology and histology specimens, respectively. Results: Ki‐67 staining was successful in 17/24 smears (71%) and 19/23 sections (83%). The correlation coefficient between the percentage of Ki‐67‐positive cells in cytological smears and in the histological sections was 0.677 (P < 0.01). These values were significantly different between histologically benign and malignant tumour groups both in cytology and histology samples (P < 0.001). The threshold value of the percentage of Ki‐67‐positive cells for distinguishing benign from malignant tumours was set at 4.85% with 90.9% sensitivity and 92.3% specificity by Receiver Operating Characteristic (ROC) curve using histopathology as the gold standard. Conclusion: Diff‐Quik‐stained cytology smears can be used to detect the presence of Ki‐67 antigen when histology sections are not available.     

Dysregulation of the Bmi‐1/p16Ink4a pathway provokes an aging‐associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16^Ink4a. Therefore, dysregulation of the Bmi‐1/p16^Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16^Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16^Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16^Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16^Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.     

The fibroblast growth factor‐2 is not essential for melanoma formation in a transgenic mouse model

Fibroblast growth factor 2 (FGF2) has been assigned a role in melanocyte proliferation and in development of human cutaneous melanoma. We have used a transgenic mouse melanoma model in combination with mice lacking mouse FGF2 to analyse the possible implication of FGF2 in melanomagenesis. Tyr::N‐ras^Q61K transgenic mice which are deficient for FGF2 and the tumor suppressors p16^INK4a and p19^ARF are hyperpigmented and develop cutaneous metastasizing melanoma, with no difference to mice wildtype for FGF2. We conclude from our data, that FGF2 is not essential for melanoma progression and metastasis.     

Long‐term repopulation of aged bone marrow stem cells using young Sca‐1 cells promotes aged heart rejuvenation

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca‐1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca‐1⁺ or Sca‐1^? cells from young (2–3 months) or aged (18–19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca‐1⁺, young Sca‐1^?, old Sca‐1⁺, and old Sca‐1^?. Four months later, expression of rejuvenation‐related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase‐related protein (DNA‐PKcs, TRF‐2) expression, whereas expression of senescence‐related genes (p16^INK4a, P19^ARF, p27^Kip1) and proteins (p16^INK4a, p27^Kip1) was decreased in Sca‐1⁺ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP^?CD31⁺) but not cardiomyocytes were the primary cell type rejuvenated by young Sca‐1⁺ cells as shown by improved proliferation, migration, and tubular formation abilities. C‐X‐C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP⁺) cells isolated from young Sca‐1⁺ chimeric hearts. Protein expression of Cxcr4, phospho‐Akt, and phospho‐FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca‐1⁺ group. Reconstitution of aged BM with young Sca‐1⁺ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.     

Restored immune cell functions upon clearance of senescence in the irradiated splenic environment

Some studies show eliminating senescent cells rejuvenate aged mice and attenuate deleterious effects of chemotherapy. Nevertheless, it remains unclear whether senescence affects immune cell function. We provide evidence that exposure of mice to ionizing radiation (IR) promotes the senescent‐associated secretory phenotype (SASP) and expression of p16^INK4a in splenic cell populations. We observe splenic T cells exhibit a reduced proliferative response when cultured with allogenic cells in vitro and following viral infection in vivo. Using p16‐3MR mice that allow elimination of p16^INK4a‐positive cells with exposure to ganciclovir, we show that impaired T‐cell proliferation is partially reversed, mechanistically dependent on p16^INK4a expression and the SASP. Moreover, we found macrophages isolated from irradiated spleens to have a reduced phagocytosis activity in vitro, a defect also restored by the elimination of p16^INK4a expression. Our results provide molecular insight on how senescence‐inducing IR promotes loss of immune cell fitness, which suggest senolytic drugs may improve immune cell function in aged and patients undergoing cancer treatment.     

Epigenetic regulation of the INK4b-ARF-INK4a locus

The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15^INK4b and p16^INK4a and a regulator of the p53 pathway, ARF. In addition ANRIL, a non-coding RNA, is also transcribed from the locus. ARF, p15^INK4b and p16^INK4a are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer’s disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs), and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.     

Outcome of SurePath™ cervical samples reported as borderline nuclear change by cytological subtype and high‐risk HPV status

N. Gupta, N. Dudding, J. Crossley, S.J. Payyappilly and J.H.F. Smith
Outcome of SurePath? cervical samples reported as borderline nuclear changes by cytological subtype and high‐risk HPV status Background: The average borderline rate in cervical cytology samples for English laboratories was 3.8% with the range being 2.0–6.8% at the time of the present study, which was undertaken in order to determine the association between different subtypes of borderline nuclear change (BNC), high‐grade cervical intraepithelial neoplasia (CIN) and high‐risk human papillomavirus (hrHPV) status. Materials and methods: Of 68 551 SurePath^TM cervical samples reported in one laboratory over a period of 2 years, 2335 (3.4%) were reported as BNC. hrHPV status was known in 1112 cases (47.6%). The outcome was known only for women with hrHPV‐positive BNC, who were recommended for colposcopy under the National Health Service Cervical Screening Programme sentinel site protocol. Women with hrHPV‐negative BNC were returned to 3‐yearly recall. The cases were subdivided into BNC, high‐grade dyskaryosis cannot be excluded (B‐HG; 105 cases); BNC with koilocytosis (B‐K; 421 cases); BNC with other features of HPV (B‐HPV; 160 cases); and BNC, not otherwise specified (B‐NOS; 426 cases) and were correlated with the histological outcome where available. Results: The study population age ranged from 23 to 65 years. Cases that tested positive for hrHPV by Qiagen HCII assay comprised 78.1%, 81.0%, 73.1% and 67.8% of B‐HG, B‐K, B‐HPV and B‐NOS categories, respectively. CIN2 or worse (CIN2+) was found in 64.6%, 10.0%, 19.7% and 20.1% of hrHPV‐positive cases of B‐HG, B‐K, B‐HPV and B‐NOS, respectively, which was significantly higher in the B‐HG category (P < 0.001) and lower in the B‐K category compared with B‐NOS (p < 0.001) and B‐HPV (p = 0.006) respectively. CIN3+ comprised 55.6%, 6.3%, 26.3% and 19.1% of biopsies in the same categories, respectively. Conclusions: Subtyping BNC is useful, especially B‐K and B‐HG, which, respectively, had the lowest and highest rates of detection of both CIN2+ and CIN3+, confirming that koilocytosis is likely to be associated with transient HPV infection. Women with B‐HG should be referred to colposcopy in the absence of HPV triage.     

Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

Central giant cell lesion of the jaws: study of CCND1 gene amplification and p16INK4a protein levels

Central giant cell lesions (CGCLs) are uncommon benign jaw lesions with uncertain etiology and a variable clinical behavior. In neoplasms, alterations in molecules involved in the G1/S checkpoint are frequently found. Loss of p16^INK4a expression or overexpression of cyclin D1 may stimulate cell proliferation. The purpose of this study was to analyze CCND1 gene amplification and the expression of p16^INK4a in CGCLs. Structural analysis of the CCND1 was performed using chromogenic in situ hybridization. Immmunohistochemistry was used to identify p16^INK4a protein levels. Statistical analysis correlated the two biomarkers with clinical behavior and between each other. Twenty-four lesions were included, being 11 aggressive and 13 non-aggressive. Moderate/high-level CCND1 amplification was found in 12 lesions. Also, immunoreactivity for p16^INK4a was present in 12 cases, mainly in mononuclear cells. There was a significantly higher level of p16^INK4a expression in mononuclear cells of non-aggressive lesions and lesions with moderate/high-level CCND1 amplification in mononuclear cells. It could be speculated that some CGCLs may develop as a true benign neoplasm. The higher expression of p16^INK4a in non-aggressive lesions and in cases with moderate/high-level CCND1 amplification may show that these molecules have a role in CGCLs.     

Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine

H. Ohsaki, E. Hirakawa, M. Nakamura, Y. Norimatsu, H. Kiyomoto and R. Haba Expression of vimentin and high‐molecular‐weight cytokeratin (clone 34ßE12) in differentiating reactive renal tubular cells from low‐grade urothelial carcinoma cells in voided urine Objective: Reactive renal tubular cells show features of an atypical repair reaction. Differentiation between reactive renal tubular cells and low‐grade urothelial carcinoma (LG‐UC) cells can therefore be a diagnostic challenge based on morphology alone. In this study, we evaluated the diagnostic utility of vimentin and a high‐molecular‐weight cytokeratin antibody (clone 34ßE12) in differentiating reactive renal tubular cells from LG‐UC. Methods: We evaluated voided urine cytology and surgical specimens from 40 patients with renal disease, and 17 patients with LG‐UC. All slides were stained with vimentin and 34ßE12. Results: In the reactive renal tubular cells in voided urine cytology, vimentin showed strong cytoplasmic staining in 39/40 (97.5%) cases, but all were negative for 34ßE12. LG‐UC cells showed positive staining for 34ßE12 in 3/17 (17.6%) cases, whereas none were positivity for vimentin. The reactive renal tubular cells of histological specimens in the renal disease group demonstrated positive for vimentin in all 40 cases and all were negative for 34ßE12. The LG‐UC group showed abnormal staining for 34ßE12 in 4/17 (23.5%) cases, whereas none were positive for vimentin. Conclusions: Vimentin expression in urine cytology can help to distinguish reactive renal tubular cells from LG‐UC. However, 34ßE12 does not appear to be a useful adjunct to distinguish these two groups in voided urine cytology.