Sufentanil alleviates cerebral ischemia-reperfusion injury by inhibiting inflammation and protecting the blood-brain barrier in rats

Stroke is a brain system disease with a high fatality rate and disability rate. About 80% of strokes are ischemic strokes. Cerebral ischemia-reperfusion injury (CIRI) caused by ischemic stroke seriously affects the prognosis of stroke patients. The purpose of this study is to investigate the effect of sufentanil (SUF) on CIRI model rats. We used middle cerebral artery occlusion (MCAO) to make the CIRI model in rats and monitored region cerebral blood flow (rCBF) to ensure that blood flow was blocked and recanalized. We used ELISA and RT-PCR to detect the expression of inflammatory factors in rat serum and brain tissue. In addition, we detected the expression of metalloproteinase (MMP) 2, MMP9 and collagen IV in brain tissues and performed Evans blue (EB) assay to determine the permeability of the blood-brain barrier (BBB). Finally, we clarified the apoptosis of brain tissue through the TUNEL staining and the detection of caspase3, Bcl2 and Bax. Various concentrations of SUF, especially 5, 10 and 25 μg/kg of SUF, all alleviated the infarct size, neurological function and brain edema of MCAO rats. SUF pretreatment also effectively reduced the expression of inflammatory cytokines in MCAO rats, including interleukin (IL)-1β, IL-4, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF)-α. In addition, SUF also inhibited MMP2 and MMP9 and promoted the expression of collagen IV, indicating that SUF attenuated the destruction of the BBB. SUF also inhibited caspase3 and Bax rats and promoted Bcl2 in MCAO rats, thus inhibiting cell apoptosis. SUF pretreatment effectively improved the neurological function and cerebral infarction of MCAO rats, inhibited excessive inflammation in rats, protected the BBB, and inhibited cell apoptosis in brain tissue.

Peroxidasin Enhances Basal Phenotype and Inhibits Branching Morphogenesis in Breast Epithelial Progenitor Cell Line D492

The human breast is composed of terminal duct lobular units (TDLUs) that are surrounded by stroma. In the TDLUs, basement membrane separates the stroma from the epithelial compartment, which is divided into an inner layer of luminal epithelial cells and an outer layer of myoepithelial cells. Stem cells and progenitor cells also reside within the epithelium and drive a continuous cycle of gland remodelling that occurs throughout the reproductive period. D492 is an epithelial cell line originally isolated from the stem cell population of the breast and generates both luminal and myoepithelial cells in culture. When D492 cells are embedded into 3D reconstituted basement membrane matrix (3D-rBM) they form branching colonies mimicking the TDLUs of the breast, thereby providing a well-suited in vitro model for studies on branching morphogenesis and breast development. Peroxidasin (PXDN) is a heme-containing peroxidase that crosslinks collagen IV with the formation of sulfilimine bonds. Previous studies indicate that PXDN plays an integral role in basement membrane stabilisation by crosslinking collagen IV and as such contributes to epithelial integrity. Although PXDN has been linked to fibrosis and cancer in some organs there is limited information on its role in development, including in the breast. In this study, we demonstrate expression of PXDN in breast epithelium and stroma and apply the D492 cell line to investigate the role of PXDN in cell differentiation and branching morphogenesis in the human breast. Overexpression of PXDN induced basal phenotype in D492 cells, loss of plasticity and inhibition of epithelial-to-mesenchymal transition as is displayed by complete inhibition of branching morphogenesis in 3D culture. This is supported by results from RNA-sequencing which show significant enrichment in genes involved in epithelial differentiation along with significant negative enrichment of EMT factors. Taken together, we provide evidence for a novel role of PXDN in breast epithelial differentiation and mammary gland development.

Associations of Single-Nucleotide Polymorphisms in Slovenian Patients with Acute Central Serous Chorioretinopathy

Central serous chorioretinopathy (CSC) is a chorioretinal disease that usually affects the middle-aged population and is characterised by a thickened choroid, retinal pigment epithelium detachment, and subretinal fluid with a tendency towards spontaneous resolution. We investigated 13 single-nucleotide polymorphisms (SNPs) in 50 Slovenian acute CSC patients and 71 healthy controls in Complement Factor H (CFH), Nuclear Receptor Subfamily 3 Group C Member 2 (NR3C2), Cadherin 5 (CDH5) Age-Related Maculopathy Susceptibility 2 (ARMS2), TNF Receptor Superfamily Member 10a (TNFRSF10A), collagen IV alpha 3 (COL4A3) and collagen IV alpha 4 (COL4A4) genes using high-resolution melt analysis. Statistical calculations revealed significant differences in genotype frequencies for CFH rs1329428 (p = 0.042) between investigated groups and an increased risk for CSC in patients with TC (p = 0.040) and TT (p = 0.034) genotype. Genotype–phenotype correlation analysis revealed that CSC patients with CC genotype in CFH rs3753394 showed a higher tendency for spontaneous CSC episode resolution at 3 months from the disease onset (p = 0.0078), which could indicate clinical significance of SNP testing in CSC patients. Bioinformatics analysis of the non-coding polymorphisms showed alterations in transcription factor binding motifs for CFH rs3753394, CDH5 rs7499886 and TNFRSF10A rs13278062. No association of collagen IV polymorphisms with CSC was found in this study.

Screening of Self-Assembling of Collagen IV Fragments into Stable Structures Potentially Useful in Regenerative Medicine

The aim of the research was to check whether it is possible to use fragments of type IV collagen to obtain, as a result of self-assembling, stable spatial structures that could be used to prepare new materials useful in regenerative medicine. Collagen IV fragments were obtained by using DMT/NMM/TosO− as a coupling reagent. The ability to self-organize and form stable spatial structures was tested by the CD method and microscopic techniques. Biological studies covered: resazurin assay (cytotoxicity assessment) on BJ, BJ-5TA and C2C12 cell lines; an alkaline version of the comet assay (genotoxicity), Biolegend Legendplex human inflammation panel 1 assay (SC cell lines, assessment of the inflammation activity) and MTT test to determine the cytotoxicity of the porous materials based on collagen IV fragments. It was found that out of the pool of 37 fragments (peptides 1–33 and 2.1–2.4) reconstructing the outer sphere of collagen IV, nine fragments (peptides: 2, 4, 5, 6, 14, 15, 25, 26 and 30), as a result of self-assembling, form structures mimicking the structure of the triple helix of native collagens. The stability of spatial structures formed as a result of self-organization at temperatures of 4 °C, 20 °C, and 40 °C was found. The application of the MST method allowed us to determine the Kd of binding of selected fragments of collagen IV to ITGα1β1. The stability of the spatial structures of selected peptides made it possible to obtain porous materials based on their equimolar mixture. The formation of the porous materials was found for cross-linked structures and the material stabilized only by weak interactions. All tested peptides are non-cytotoxic against all tested cell lines. Selected peptides also showed no genotoxicity and no induction of immune system responses. Research on the use of porous materials based on fragments of type IV collagen, able to form stable spatial structures as scaffolds useful in regenerative medicine, will be continued.

Coupling adipose tissue architecture and metabolism via cytoophidia

ABSTRACTTissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here, we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Interestingly, loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, leads to downregulated Collagen-Integrin signaling, weakened adipocyte adhesion, and defective adipose architecture. Strikingly, CTPS specifically binds with Integrin subunit α2, which influences Integrin function and Collagen IV deposition. cytoophidia promote Collagen IV mRNA expression and thus its extracellular deposition to strengthen adipocyte adhesion. Remarkably, Collagen IV-Integrin signaling reciprocally regulates cytoophidium formation at a post-translational level. Together, we demonstrate that a positive feedback signaling loop containing both cytoophidia and Integrin adhesion complex couples tissue architecture and metabolism in the fly adipose.

Anti-Aging β-Klotho Gene-Activated Scaffold Promotes Rejuvenative Wound Healing Response in Human Adipose-Derived Stem Cells

Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene β-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-β3 and TGF-β1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the β-Klotho gene-activated scaffold offers controlled activation of ADSCs’ regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-β3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the β-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.

Liposomal Nanocarriers Designed for Sub-Endothelial Matrix Targeting under Vascular Flow Conditions

Vascular interventions result in the disruption of the tunica intima and the exposure of sub-endothelial matrix proteins. Nanoparticles designed to bind to these exposed matrices could provide targeted drug delivery systems aimed at inhibiting dysfunctional vascular remodeling and improving intervention outcomes. Here, we present the progress in the development of targeted liposomal nanocarriers designed for preferential collagen IV binding under simulated static vascular flow conditions. PEGylated liposomes (PLPs), previously established as effective delivery systems in vascular cells types, served as non-targeting controls. Collagen-targeting liposomes (CT-PLPs) were formed by conjugating established collagen-binding peptides to modified lipid heads via click chemistry (CTL), and inserting them at varying mol% either at the time of PLP assembly or via micellar transfer. All groups included fluorescently labeled lipid species for imaging and quantification. Liposomes were exposed to collagen IV matrices statically or via hemodynamic flow, and binding was measured via fluorometric analyses. CT-PLPs formed with 5 mol% CTL at the time of assembly demonstrated the highest binding affinity to collagen IV under static conditions, while maintaining a nanoparticle characterization profile of ~50 nm size and a homogeneity polydispersity index (PDI) of ~0.2 favorable for clinical translation. When liposomes were exposed to collagen matrices within a pressurized flow system, empirically defined CT-PLPs demonstrated significant binding at shear stresses mimetic of physiological through pathological conditions in both the venous and arterial architectures. Furthermore, when human saphenous vein explants were perfused with liposomes within a closed bioreactor system, CT-PLPs demonstrated significant ex vivo binding to diseased vascular tissue. Ongoing studies aim to further develop CT-PLPs for controlled targeting in a rodent model of vascular injury. The CT-PLP nanocarriers established here show promise as the framework for a spatially controlled delivery platform for future application in targeted vascular therapeutics.

Immunomorphological features of female patient’s skin with androgenetic alopecia in the treatment of platelet-rich plasma in combination with topical minoxidil 2 % lotion

The aim. The research was provided to obtain immunohistochemical changes in scalp biopsies of female patients with androgenetic alopecia (AGA) I-II stages according to the Ludwig scale and to detect possible changes after usage of platelet-rich plasma (PRP) injections in combination with topical minoxidil 2 % lotion. Materials and methods. Skin biopsies of 30 patients with AGA were examined by immunohistochemistry before and after treatment, which lasted for 3 months and included 3 PRP sessions (once per month) and topical application of minoxidil 2 % lotion twice daily. Results. It was found that AGA is accompanied by inflammatory perifollicular infiltration by T-lymphocytes CD3 +, CD4 + and CD8 +, macrophages (CD68 +); imbalance of growth polypeptides VEGF, TGF-β1, EGFR; accumulation of oxidative stress enzymes eNOS and iNOS; accumulation of pathological fraction of Collagen IV. The use of platelet-rich plasma in combination with topical 2 % minoxidil as AGA therapy leads to the normalization of immunohistochemical parameters of the skin, which indicates the possibility of its use for long-term therapeutic effect. Conclusions. This study supplemented the understanding of the pathogenesis of AGA and serves as the basis for improving treatment regimens for this pathology. But more research is required to further study the pathomorphology of androgenetic alopecia and to standardize the technique of using platelet-rich plasma in patients with this disease.