The aged extracellular matrix and the profibrotic role of senescence-associated secretory phenotype.

Idiopathic pulmonary fibrosis (IPF) is an irreversible and fatal lung disease that is primarily found in the elderly population, and several studies have demonstrated that aging is the major risk factor for IPF. IPF is characterized by the presence of apoptosis-resistant, senescent fibroblasts that generate an excessively stiff extracellular matrix (ECM). The ECM profoundly affects cellular functions and tissue homeostasis, and an aberrant ECM is closely associated with the development of lung fibrosis. Aging progressively alters ECM components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction through the expression of factors linked to a senescence-associated secretary phenotype (SASP). There is growing evidence that SASP factors affect various cell behaviors and influence ECM turnover in lung tissue through autocrine and/or paracrine signaling mechanisms. Since life expectancy is increasing worldwide, it is important to elucidate how aging affects ECM dynamics and turnover via SASP and thereby promotes lung fibrosis. In this review, we will focus on the molecular properties of SASP and its regulatory mechanisms. Furthermore, the pathophysiological process of ECM remodeling by SASP factors and the influence of an altered ECM from aged lungs on the development of lung fibrosis will be highlighted. Finally, recent attempts to target ECM alteration and senescent cells to modulate fibrosis will be introduced.NEW & NOTEWORTHY Aging is the most prominent nonmodifiable risk factor for various human diseases including Idiopathic pulmonary fibrosis. Aging progressively alters extracellular matrix components and is associated with the accumulation of senescent cells that promote age-related tissue dysfunction. In this review, we will discuss the pathological impact of aging and senescence on lung fibrosis via senescence-associated secretary phenotype factors and potential therapeutic approaches to limit the progression of lung fibrosis.

Independent role of caspases and Bik in augmenting influenza A virus replication in airway epithelial cells and mice.

Caspases and poly (ADP-ribose) polymerase 1 (PARP1) have been shown to promote influenza A virus (IAV) replication. However, the relative importance and molecular mechanisms of specific caspases and their downstream substrate PARP1 in regulating viral replication in airway epithelial cells (AECs) remains incompletely elucidated. Here, we targeted caspase 2, 3, 6, and PARP1 using specific inhibitors to compare their role in promoting IAV replication. Inhibition of each of these proteins caused significant decline in viral titer, although PARP1 inhibitor led to the most robust reduction of viral replication. We previously showed that the pro-apoptotic protein Bcl-2 interacting killer (Bik) promotes IAV replication in the AECs by activating caspase 3. In this study, we found that as compared with AECs from wild-type mice, bik-deficiency alone resulted in ~ 3 logs reduction in virus titer in the absence of treatment with the pan-caspase inhibitor (Q-VD-Oph). Inhibiting overall caspase activity using Q-VD-Oph caused additional decline in viral titer by ~ 1 log in bik-/- AECs. Similarly, mice treated with Q-VD-Oph were protected from IAV-induced lung inflammation and lethality. Inhibiting caspase activity diminished nucleo-cytoplasmic transport of viral nucleoprotein (NP) and cleavage of viral hemagglutinin and NP in human AECs. These findings suggest that caspases and PARP1 play major roles to independently promote IAV replication and that additional mechanism(s) independent of caspases and PARP1 may be involved in Bik-mediated IAV replication. Further, peptides or inhibitors that target and block multiple caspases or PARP1 may be effective treatment targets for influenza infection.

MAPKAPK2-centric transcriptome profiling reveals its major role in governing molecular crosstalk of IGFBP2, MUC4, and PRKAR2B during HNSCC pathogenesis.

Transcriptome analysis of head and neck squamous cell carcinoma (HNSCC) has been pivotal to comprehending the convoluted biology of HNSCC tumors. MAPKAPK2 or MK2 is a critical modulator of the mRNA turnover of crucial genes involved in HNSCC progression. However, MK2-centric transcriptome profiles of tumors are not well known. This study delves into HNSCC progression with MK2 at the nexus to delineate the biological relevance and intricate crosstalk of MK2 in the tumor milieu. We performed next-generation sequencing-based transcriptome profiling of HNSCC cells and xenograft tumors to ascertain mRNA expression profiles in MK2-wild type and MK2-knockdown conditions. The findings were validated using gene expression assays, immunohistochemistry, and transcript turnover studies. Here, we identified a pool of crucial MK2-regulated candidate genes by annotation and differential gene expression analyses. Regulatory network and pathway enrichment revealed their significance and involvement in the HNSCC pathogenesis. Additionally, 3'-UTR-based filtering recognized important MK2-regulated downstream target genes and validated them by nCounter gene expression assays. Finally, immunohistochemistry and transcript stability studies revealed the putative role of MK2 in regulating the transcript turnover of IGFBP2, MUC4, and PRKAR2B in HNSCC. Conclusively, MK2-regulated candidate genes were identified in this study, and their plausible involvement in HNSCC pathogenesis was elucidated. These genes possess investigative values as targets for diagnosis and therapeutic interventions for HNSCC.

B-cell lymphoma-2 family proteins-activated proteases as potential therapeutic targets for influenza A virus and severe acute respiratory syndrome coronavirus-2: Killing two birds with one stone?

The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to a global health emergency. There are many similarities between SARS-CoV-2 and influenza A virus (IAV); both are single-stranded RNA viruses infecting airway epithelial cells and have similar modes of replication and transmission. Like IAVs, SARS-CoV-2 infections poses serious challenges due to the lack of effective therapeutic interventions, frequent appearances of new strains of the virus, and development of drug resistance. New approaches to control these infectious agents may stem from cellular factors or pathways that directly or indirectly interact with viral proteins to enhance or inhibit virus replication. One of the emerging concepts is that host cellular factors and pathways are required for maintaining viral genome integrity, which is essential for viral replication. Although IAVs have been studied for several years and many cellular proteins involved in their replication and pathogenesis have been identified, very little is known about how SARS-CoV-2 hijacks host cellular proteins to promote their replication. IAV induces apoptotic cell death, mediated by the B-cell lymphoma-2 (Bcl-2) family proteins in infected epithelia, and the pro-apoptotic members of this family promotes viral replication by activating host cell proteases. This review compares the life cycle and mode of replication of IAV and SARS-CoV-2 and examines the potential roles of host cellular proteins, belonging to the Bcl-2 family, in SARS-CoV-2 replication to provide future research directions.

AMPK-driven Macrophage Responses Are Autophagy Dependent in Experimental Bronchopulmonary Dysplasia.

The pathogenesis of bronchopulmonary dysplasia (BPD) remains incompletely understood. Recent studies suggest insufficient AMP-activated protein kinase (AMPK) activation as a potential cause of impaired autophagy in rodent and nonhuman primate models of BPD. Impaired autophagy is associated with enhanced inflammatory signaling in alveolar macrophages (AMs) and increased severity of murine BPD induced by neonatal hyperoxia exposure. The goal of this study was to determine the role of autophagy and AMPK activation in macrophage responses in murine BPD. C57BL/6J mice were exposed to neonatal hyperoxia starting on postnatal day (P)1 and treated with the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) between P3 and P6. Mice were euthanized on P7, and markers of AMPK activation and autophagy were assessed by immunoblotting. Alveolarization was assessed using radial alveolar counts, mean linear intercept measurements, and quantification of alveolar septal myofibroblasts. Relative mRNA expression of M1-like and M2-like genes was assessed in AMs isolated from BAL fluid from wild-type, LysMCre--Becn1fl/fl, and LysMCre+-Becn1fl/fl mice after neonatal hyperoxia exposure. AICAR treatment resulted in AMPK activation and induction of autophagic activity in whole-lung and BAL cell lysates and attenuated hyperoxia-induced alveolar simplification in neonatal lungs. AICAR-treated control but not Beclin1-deficient AMs demonstrated significantly decreased expression of M1-like markers and significantly increased expression of M2-like markers. In conclusion, pharmacologic activation of AMPK by AICAR resulted in induction of autophagy and played a protective role, at least in part, through attenuation of proinflammatory signaling in AMs via autophagy-dependent mechanisms in a murine model of BPD.

Comparative analysis of ACE2 protein expression in rodent, non-human primate, and human respiratory tract at baseline and after injury: A conundrum for COVID-19 pathogenesis.

Angiotensin converting enzyme 2 (ACE2) is the putative functional receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current literature on the abundance and distribution of ACE2 protein in the human respiratory tract is controversial. We examined the effect of age and lung injury on ACE2 protein expression in rodent and non-human primate (NHP) models. We also examined ACE2 expression in human tissues with and without coronavirus disease 19 (COVID-19). ACE2 expression was detected at very low levels in preterm, but was absent in full-term and adult NHP lung homogenates. This pattern of ACE2 expression contrasted with that of transmembrane protease serine type 2 (TMPRSS2), which was significantly increased in full-term newborn and adult NHP lungs compared to preterm NHP lungs. ACE2 expression was not detected in NHP lungs with cigarette smoke-induced airway disease or bronchopulmonary dysplasia. Murine lungs lacked basal ACE2 immunoreactivity, but responded to hyperoxia, bacterial infection, and allergen exposure with new ACE2 expression in bronchial epithelial cells. In human specimens, robust ACE2 immunoreactivity was detected in ciliated epithelial cells in paranasal sinus specimens, while ACE2 expression was detected only in rare type 2 alveolar epithelial cells in control lungs. In autopsy specimens from patients with COVID-19 pneumonia, ACE2 was detected in rare ciliated epithelial and endothelial cells in the trachea, but not in the lung. There was robust expression of ACE2 expression in F344/N rat nasal mucosa and lung specimens, which authentically recapitulated the ACE2 expression pattern in human paranasal sinus specimens. Thus, ACE2 protein expression demonstrates a significant gradient between upper and lower respiratory tract in humans and is scarce in the lung. This pattern of ACE2 expression supports the notion of sinonasal epithelium being the main entry site for SARS-CoV-2 but raises further questions on the pathogenesis and cellular targets of SARS-CoV-2 in COVID-19 pneumonia.

Impaired Autophagic Activity Contributes to the Pathogenesis of Bronchopulmonary Dysplasia. Evidence from Murine and Baboon Models.

Bronchopulmonary dysplasia (BPD) is a common and serious complication associated with preterm birth. The pathogenesis of BPD is incompletely understood, and there is an unmet clinical need for effective treatments. The role of autophagy as a potential cytoprotective mechanism in BPD remains to be fully elucidated. In the present study, we investigated the role and regulation of autophagy in experimental models of BPD. Regulation and cellular distribution of autophagic activity during postnatal lung development and in neonatal hyperoxia-induced lung injury (nHILI) were assessed in the autophagy reporter transgenic GFP-LC3 (GFP-microtubule-associated protein 1A/1B-light chain 3) mouse model. Autophagic activity and its regulation were also examined in a baboon model of BPD. The role of autophagy in nHILI was determined by assessing lung morphometry, injury, and inflammation in autophagy-deficient Beclin 1 heterozygous knockout mice (Becn1+/-). Autophagic activity was induced during alveolarization in control murine lungs and localized primarily to alveolar type II cells and macrophages. Hyperoxia exposure of neonatal murine lungs and BPD in baboon lungs resulted in impaired autophagic activity in association with insufficient AMPK (5'-AMP-activated protein kinase) and increased mTORC1 (mTOR complex 1) activation. Becn1+/- lungs displayed impaired alveolarization, increased alveolar septal thickness, greater neutrophil accumulation, and increased IL-1ß concentrations when exposed to nHILI. Becn1+/- alveolar macrophages isolated from nHILI-exposed mice displayed increased expression of proinflammatory genes. In conclusion, basal autophagy is induced during alveolarization and disrupted during progression of nHILI in mice and BPD in baboons. Becn1+/- mice are more susceptible to nHILI, suggesting that preservation of autophagic activity may be an effective protective strategy in BPD.

MAPKAPK2 plays a crucial role in the progression of head and neck squamous cell carcinoma by regulating transcript stability.

BACKGROUND: Head and neck squamous-cell carcinoma (HNSCC) ranks sixth among cancers worldwide. Though several molecular mechanisms of tumor initiation and progression of HNSCC are known, others remain unclear. Significance of p38/MAPKAPK2 (Mitogen-activated protein kinase-activated protein kinase-2) pathway in cell stress and inflammation is well established and its role in tumor development is being widely studied. METHODS: We have elucidated the role of MAPKAPK2 (MK2) in HNSCC pathogenesis using clinical tissue samples, MK2-knockdown (MK2KD) cells and heterotropic xenograft mice model. RESULTS: In patient-derived tissue samples, we observed that MK2 is reproducibly overexpressed. Increased stability of cyclin-dependent kinase inhibitor 1B (p27), mitogen-activated protein kinase phosphatase-1 (MKP-1) transcripts and decreased half-life of tumor necrosis factor-alpha (TNF-α) and vascular endothelial growth factor (VEGF) transcripts in MK2KD cells suggests that MK2 regulates their transcript stability. In vivo xenograft experiments established that knockdown of MK2 attenuates course of tumor progression in immunocompromised mice. CONCLUSION: Altogether, MK2 is responsible for regulating the transcript stability and is functionally important to modulate HNSCC pathogenesis.

MAPKAPK2: the master regulator of RNA-binding proteins modulates transcript stability and tumor progression.

The p38 mitogen-activated protein kinase (p38MAPK) pathway has been implicated in a variety of pathological conditions including inflammation and metastasis. Post-transcriptional regulation of genes harboring adenine/uridine-rich elements (AREs) in their 3'-untranslated region (3'-UTR) is controlled by MAPK-activated protein kinase 2 (MAPKAPK2 or MK2), a downstream substrate of the p38MAPK. In response to diverse extracellular stimuli, MK2 influences crucial signaling events, regulates inflammatory cytokines, transcript stability and critical cellular processes. Expression of genes involved in these vital cellular cascades is controlled by subtle interactions in underlying molecular networks and post-transcriptional gene regulation that determines transcript fate in association with RNA-binding proteins (RBPs). Several RBPs associate with the 3'-UTRs of the target transcripts and regulate their expression via modulation of transcript stability. Although MK2 regulates important cellular phenomenon, yet its biological significance in tumor progression has not been well elucidated till date. In this review, we have highlighted in detail the importance of MK2 as the master regulator of RBPs and its role in the regulation of transcript stability, tumor progression, as well as the possibility of use of MK2 as a therapeutic target in tumor management.

Oral Insulin: Myth or Reality.

BACKGROUND: Diabetes Mellitus (DM) is a disorder of glucose metabolism marked by hyperglycemia, glycosuria, hyperlipidemia, negative nitrogen balance and ketonaemia. DM is a major healthcare problems today and its treatment costs billions of dollars worldwide annually. The cases of diabetes have increased rapidly in recent years throughout the world. Currently, for the management of Type-1 Diabetes Mellitus (TIDM), Multiple Daily Insulin (MDI) injections is the most popular treatment. Oral administration of insulin is the most suitable and attractive as compared to subcutaneous route but unfortunately cannot be utilized for the administration of peptides and proteins due to poor epithelial permeability and enzymatic degradation within the gastrointestinal tract. Since many years, extensive research has been carried out to explore the potential ways of insulin administration based on novel methods such as liposome, microsphere, nanoparticle, mouth dissolving strips, sprays exploiting oral and pulmonary route. These next generation efficient therapies for T1DM may help to improve the quality of life of diabetic patients especially in Insulin Dependent Diabetes Mellitus (IDDM). CONCLUSION: This review emphasizes on the most recent progress made in the development of oral insulin delivery formulations, and focuses on key lessons and implications from studies undertaken till date with the oral insulin formulations. Further, this review analyzes effectiveness of the advancements, applications and limitations of the technologies in delivering insulin to the targeted site through oral administration.

Cytocompatible Anti-microbial Dressings of Syzygium cumini Cellulose Nanocrystals Decorated with Silver Nanoparticles Accelerate Acute and Diabetic Wound Healing.

The ever increasing incidences of non-healing skin wounds have paved way for many efforts on the convoluted process of wound healing. Unfortunately, the lack of relevance and success of modern wound dressings in healing of acute and diabetic wounds still remains a matter of huge concern. Here, an in situ three step approach was embraced for the development of nanocomposite (NCs) dressings by impregnating silver nanoparticles (AgNPs) onto a matrix of cellulose nanocrystals (CNCs) isolated from Syzygium cumini leaves using an environmental friendly approach. Topical application of NCs (ointments and strips) on acute and diabetic wounds of mice documented enhanced tissue repair (~99% wound closure) via decrease in inflammation; increase in angiogenesis, collagen deposition, and rate of neo-epithelialization that ultimately led to formation of aesthetically sound skin in lesser time than controls. Due to the synergistic action of CNCs (having high water uptake capacity) and AgNPs (anti-microbial agents), NCs tend to increase the expression of essential growth factors (FGF, PDGF and VEGF) and collagen while decreasing the pro-inflammatory factors (IL-6 and TNF-α) at the same time, thus accelerating healing. The results suggested the potential of these developed anti-microbial, cytocompatible and nanoporous NCs having optimized AgNPs concentration as ideal dressings for effective wound management.

Picrorhiza kurroa Enhances ß-Cell Mass Proliferation and Insulin Secretion in Streptozotocin Evoked ß-Cell Damage in Rats.

Autoimmune destruction of insulin producing pancreatic ß-cells leads to insulin insufficiency and hyperglycemia in type 1 diabetes mellitus. Regeneration of ß-cells is one of the proposed treatment for type 1 diabetes and insulin insufficiency. Picrorhiza kurroa is a medicinal herb and is traditionally being used for the treatment of various diseases. Previous studies reported the hypoglycemic potential of P. kurroa. However, its potential role in ß-cell induction in insulin secretion have not been fully investigated. Here, we characterized the hydro alcoholic extract of P. kurroa rhizome (PKRE) and further studied its ß-cell regeneration and induction of insulin secretion potential in streptozotocin (STZ) induced diabetic rats as well as in insulin producing Rin5f cells. 1H-NMR revealed the presence of more than thirty metabolites including picroside I and II in PKRE. Further, we found that PKRE treatment (100 and 200 mg/kg dose for 30 days) significantly (p ≤ 0.05) protected the pancreatic ß-cells against streptozotocin (STZ) evoked damage and inhibited the glucagon receptor expression (Gcgr) in hepatic and renal tissues. It significantly (p ≤ 0.05) enhanced the insulin expression and aids in proliferation of insulin producing Rin5f cells with elevated insulin secretion. Furthermore it significantly (p ≤ 0.05) increased insulin mediated glucose uptake in 3T3L1 and L6 cells. On the contrary, in diabetic rats, PKRE significantly (p ≤ 0.05) decreased high blood glucose and restored the normal levels of serum biochemicals. Altogether, our results showed that PKRE displayed ß-cell regeneration with enhanced insulin production and antihyperglycemic effects. PKRE also improves hepatic and renal functions against oxidative damage.

In vivo diabetic wound healing potential of nanobiocomposites containing bamboo cellulose nanocrystals impregnated with silver nanoparticles.

In diabetes, hyperglycemic state immensely hinders the wound healing. Here, nanobiocomposites (NCs) developed by impregnation of in situ prepared silver nanoparticles in the matrix of bamboo cellulose nanocrystals were investigated for their ability to hasten the progress of healing events in streptozotocin induced diabetic mice model. Wounds treated with topically applied NCs (hydrogels) showed full recovery (98-100%) within 18days post wounding in contrast to the various control groups where incomplete healing (88-92%) was noticed. Biochemical estimations documented a marked decrease in the levels of pro-inflammatory cytokines IL-6 and TNF-α leading to decreased inflammation in NCs treated mice. Significantly increased expression of collagen and growth factors (FGF, PDGF, VEGF) upon NCs treatment resulted in improved re-epithelialization, vasculogenesis and collagen deposition as compared to control groups. Hence, developed nanobiocomposites showcased potential to serve as highly effective and biocompatible wound dressings for diabetic patients.

Sustained delivery of BSA/HSA from biocompatible plant cellulose nanocrystals for in vitro cholesterol release from endothelial cells.

Nanocomposites of plant cellulose nanocrystals (CNCs) were developed by binding model proteins BSA and HSA onto CNCs by physical adsorption and chemical conjugation methods The spectroscopy and microscopy studies confirmed the protein binding onto CNCs. Phosphate buffer saline (pH=4.0, 7.4) and simulated gastric and intestinal fluids (SGF/SIF; pH=1.1/6.5) showed maximum protein release of ∼62% over a period of time. The released proteins were found to retain both structural integrity as well as≥90% of bioactivity. Further, these cytocompatible nanocomposites showed ∼58-85% cholesterol release from HUVEC whereas no selectivity was observed for HCAEC. It is speculated that due to the presence of combination of shuttles (albumins) and sinks (CNCs and albumins), these prepared nanocomposites with increased cholesterol effluxing ability may serve as a potential candidate for future biomedical applications in pharmaceuticals.

HIF-1 in cancer therapy: two decade long story of a transcription factor.

BACKGROUND: Oxygen (O2) homeostasis is an indispensable requirement of eukaryotes. O2 concentration in cellular milieu is defined as normoxia (∼21% O2), physoxia (∼1-13% O2) or hypoxia (∼0.1-1% O2). Hypoxia, a striking micro-environmental feature in tumorigenesis, is countered by tumor cells via induction of O2 governed transcription factor, hypoxia inducible factor-1 (HIF-1). Post discovery, HIF-1 has emerged as a promising anticancer therapeutic target during the last two decades. Recent reports have highlighted that enhanced levels of HIF-1 correlate with tumor metastasis leading to poor patient prognosis. MATERIAL AND METHODS: A systematic search in PubMed and SciFinder for the literature on HIF-1 biology and therapeutic importance in cancer was carried out. RESULTS: This review highlights the initial description as well as the recent insights into HIF-1 biology and regulation. We have focused on emerging data regarding varied classes of HIF-1 target genes affecting various levels of crosstalk among tumorigenic pathways. We have emphasized on the fact that HIF-1 acts as a networking hub coordinating activities of multiple signaling molecules influencing tumorigenesis. Emerging evidences indicate role of many HIF-induced proteomic and genomic alterations in malignant progression by mediating a myriad of genes stimulating angiogenesis, anaerobic metabolism and survival of cancer cells in O2-deficient microenvironment. CONCLUSIONS: Better understanding of the crucial role of HIF-1 in carcinogenesis could offer promising new avenues to researchers and aid in elucidating various open issues regarding the use of HIF-1 as an anticancer therapeutic target. In spite of large efforts in this field, many questions still remain unanswered. Hence, future investigations are necessary to devise, assess and refine methods for translating previous research efforts into novel clinical practices in cancer treatment.

In situ functionalized nanobiocomposites dressings of bamboo cellulose nanocrystals and silver nanoparticles for accelerated wound healing.

An innovative approach was adopted where in situ synthesized silver nanoparticles (AgNPs) from leaf extract mediated reduction of AgNO3 were simultaneously impregnated into the matrix of cellulose nanocrystals (CNCs) isolated from Dendrocalamus hamiltonii and Bambusa bambos leaves, for formation of nanobiocomposites (NCs) in film and ointment forms. Here, use of plant CNCs was chosen as an alternate to bacterial cellulose for wound dressings. NCs possessing water absorption capacity and strong antibacterial activity showed synergistic effect on in vivo skin wound healing and documented faster and significant wound closure in treated mice. NCs exhibited lesser inflammation and early vasculogenesis at day 3 coupled with increased fibroblasts and collagen content at day 8 leading to faster neo-epithelization by day 14. Highly effective, biocompatible, and easy to apply NCs wound dressings (ointment and films) containing low amounts of Ag (0.05±0.01wt%) are potential candidates for effective skin repair.