Growth Factor Secretion Research Articles

The role of circulating neutrophils in the progression of kidney cancer

Introduction. Currently, the question of the role of neutrophils in the progression of kidney cancer remains relevant. Neutrophils are capable of exhibiting protumor properties through the secretion of cytokines, chemokines, and growth factors, which is determined by the expression of genes for these molecules. And the functional heterogeneity of neutrophils is characterized by differences in gene expression patterns.Aim. To assess the role of circulating neutrophils in the progression of kidney cancer.Materials and methods. In circulating neutrophils of patients with verified clear cell kidney cancer at stages I–III according to Tumor, Nodus and Metastasis (TNM) (n = 88) before surgical treatment and conditionally healthy donors (control group) (n = 20), the expression of NGAL genes was determined using quantitative reverse transcription polymerase chain reaction, MMP-13 and VEGF-A.Results. There was an increase in NGAL gene expression in circulating neutrophils (p = 0.05) at the initial stage and a decrease in it at advanced stages of kidney cancer (p = 0.03). High expression of the MMP-13 gene by circulating neutrophils was detected at all stages of kidney cancer relative to control values (at stage I p = 0.005; at stage II p = 0.003; at stage III p = 0.0008). A significant direct correlation was observed between the expression of the NGAL and MMP-13 genes in neutrophils at stage I kidney cancer (r = 0.696; p = 0.003). In the group of patients with kidney cancer, a direct correlation was found between the expression of the NGAL and VEGF-A genes (r = 0.322; p = 0.049). A multivariable Cox regression model for disease-free survival revealed the predictive value of VEGF-A and NGAL genes expression in circulating neutrophils. With an increase in the expression of the VEGF-A and NGAL genes in neutrophils by 1 unit, the risk of metastases increases by 0.80 (0.65–0.99; p = 0.043) and 1.42 (1.01–2.00; p = 0.046) times, respectively. The Kaplan–Meier analysis of disease-free survival in patients with kidney cancer showed the influence of NGAL expression in circulating neutrophils on progression-free time. In the group of patients with high NGAL expression, the median follow-up was 31.7 months, and in the group with low NGAL expression – more than 36 months (log-rank-test; p = 0.017).Conclusion. Thus, the data obtained suggest that circulating neutrophils play a leading role in the progression of kidney cancer. The level of expression of NGAL in circulating neutrophils can be used to predict the relapse-free period in patients with kidney cancer.

Curcumin prevents high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation via mitigating intracellular free radicals and TGF-β secretion

Diabetic kidney disease (DKD) is a leading cause of kidney failure. However, the involvement of renal fibroblasts and their communications with renal epithelial cells during DKD remain poorly understood. We investigated the potential role of renal proximal tubular epithelial cells (PTECs) in renal fibroblast activation that might lead to DKD. Additionally, the protective effects of curcumin, a known antioxidant, against renal fibroblast activation induced by high glucose-treated PTECs were investigated. Secretome was collected from HK-2 PTECs under normal glucose, high glucose, high glucose pretreated/cotreated with curcumin, or osmotic control condition for 24 h. Such secretome was then used to treat BHK-21 renal fibroblasts for 24 h. BHK-21 cells treated with high glucose-induced secretome had increased levels of fibroblast activation markers, including spindle index, F-actin, α-smooth muscle actin (α-SMA), fibronectin, collagen I, matrix metalloproteinase-2 (MMP-2) and MMP-9, as compared with normal glucose and osmotic control conditions. However, all these increases were successfully mitigated by curcumin. In addition, high glucose markedly increased intracellular reactive oxygen species (ROS) and transforming growth factor-β (TGF-β) secretion, but did not affect the secretion of platelet-derived growth factor A (PDGFA) and interleukin-1β (IL-1β), in HK-2 renal cells as compared with normal glucose and osmotic control conditions. Both intracellular ROS and secreted TGF-β levels were successfully mitigated by curcumin. Therefore, curcumin prevents the high glucose-induced stimulatory effects of renal cell secretome on fibroblast activation, at least in part, via mitigating intracellular ROS and TGF-β secretion.

USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Intramuscular injection of mesenchymal stem cells augments basal muscle protein synthesis after bouts of resistance exercise in male mice.

Skeletal muscle mass is critical for activities of daily living. Resistance training maintains or increases muscle mass, and various strategies maximize the training adaptation. Mesenchymal stem cells (MSCs) are multipotent cells with differential potency in skeletal muscle cells and the capacity to secrete growth factors. However, little is known regarding the effect of intramuscular injection of MSCs on basal muscle protein synthesis and catabolic systems after resistance training. Here, we measured changes in basal muscle protein synthesis, the ubiquitin-proteasome system, and autophagy-lysosome system-related factors after bouts of resistance exercise by intramuscular injection of MSCs. Mice performed three bouts of resistance exercise (each consisting of 50 maximal isometric contractions elicited by electrical stimulation) on the right gastrocnemius muscle every 48 h, and immediately after the first bout, mice were intramuscularly injected with either MSCs (2.0 × 106 cells) labeled with green fluorescence protein (GFP) or vehicle only placebo. Seventy-two hours after the third exercise bout, GFP was detected only in the muscle injected with MSCs with concomitant elevation of muscle protein synthesis. The injection of MSCs also increased protein ubiquitination. These results suggest that the intramuscular injection of MSCs augmented muscle protein turnover at the basal state after consecutive resistance exercise.

Single dose human perinatal stem cells accelerate healing of cold-induced rat burn wound.

Temporal phases of wound healing and their corresponding healing factors are essential in wound regeneration. Mesenchymal stem cells (MSCs)accelerate wound healing via their paracrine secretions by enhancing cell migration, angiogenesis, and reducing inflammation. This study evaluated the local therapeutic effect of human umbilical cord MSCs (hUCMSCs)in the healing of cold-induced burn wounds. An in vitro wound (scratch) was developed in rat skin fibroblasts. The culture was maintained in the conditioned medium (CM)which was prepared by inducing an artificial wound in hUCMSCs in a separate experiment. Treated fibroblasts were analyzed for the gene expression profile of healing mediators involved in wound closure. Findings revealed enhanced cell migration and increased levels of healing mediators in the treated fibroblasts relative to the untreated group. Cold-induced burn wounds were developed in Wistar rats, followed by a single injection of hUCMSCs. Wound healing pattern was examined based on the healing phases: hemostasis/inflammation (Days 1, 3), cell proliferation (Day 7), and remodeling (Day 14). Findings exhibited enhanced wound closure in the treated wound. Gene expression, histological, and immunohistochemical analyses further confirmed enhanced wound regeneration after hUCMSC transplantation. Temporal gene expression profile revealed that the level of corresponding cytokines was substantially increased in the treated wound as compared with the control, indicating improvement in the processes of angiogenesis and remodeling, and a substantial reduction in inflammation. Histology revealed significant collagen formation along with regenerated skin layers and appendages, whereas immunohistochemistry exhibited increased neovascularization during remodeling. Leukocyte infiltration was also suppressed in the treated group. Overall findings demonstrate that a single dose of hUCMSCs enhances wound healing in vivo, and their secreted growth factors accelerate cell migration in vitro.

The Effect of Salvianolic Acid A on Tumor-Associated Macrophage Polarization and Its Mechanisms in the Tumor Microenvironment of Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with a high degree of malignancy and poor prognosis. Tumor-associated macrophages (TAMs) have been identified as significant contributors to the growth and metastasis of TNBC through the secretion of various growth factors and chemokines. Salvianolic acid A (SAA) has been shown to have anti-cancer activities. However, the potential activity of SAA on re-polarized TAMs remains unclear. As there is a correlation between the TAMs and TNBC, this study investigates the effect of SAA on TAMs in the TNBC microenvironment. For that purpose, M2 TAM polarization was induced by two kinds of TNBC-conditioned medium (TNBC-TCM) in the absence or presence of SAA. The gene and protein expression of TAM markers were analyzed by qPCR, FCM, IF, ELISA, and Western blot. The protein expression levels of ERK and p-ERK in M2-like TAMs were analyzed by Western blot. The migration and invasion properties of M2-like TAMs were analyzed by Transwell assays. Here, we demonstrated that SAA increased the expression levels of CD86, IL-1β, and iNOS in M2-like TAMs and, conversely, decreased the expression levels of Arg-1 and CD206. Moreover, SAA inhibited the migration and invasion properties of M2-like TAMs effectively and decreased the protein expression of TGF-β1 and p-ERK in a concentration-dependent manner, as well as TGF-β1 gene expression and secretion. Our current findings for the first time demonstrated that SAA inhibits macrophage polarization to M2-like TAMs by inhibiting the ERK pathway and promotes M2-like TAM re-polarization to the M1 TAMs, which may exert its anti-tumor effect by regulating M1/M2 TAM polarization. These findings highlight SAA as a potential regulator of M2 TAMs and the possibility of utilizing SAA to reprogram M2 TAMs offers promising insights for the clinical management of TNBC.

Abstract 5456: Identifying the role of tissue factor in osteosarcoma metastasis

Abstract Patients with osteosarcoma who develop metastasis face very poor prognoses. Even when treated with the most effective medical and surgical interventions available, about forty percent of osteosarcoma patients will eventually succumb to lung metastasis. Despite the relevance of metastasis to clinical outcomes, we understand little about the mechanisms that drive these tumor cells into the lung and facilitate their metastatic growth. Consequently, there are no approved antimetastatic agents for osteosarcoma and outcomes have not improved in more than 40 years. Tissue factor is a tightly regulated physiologic initiator of coagulation with functions in inflammation, cytokinesis, angiogenesis, apoptosis, and growth factor secretion. These functions are divided between two separate pathways: initiation of the coagulation cascade via activation of factors VII and X, and propagation of cell signaling pathways via PAR activation. It is aberrantly expressed in many cancers and has been associated with metastatic behavior and inferior outcomes by affecting metastatic vascularity, growth, and migration. Osteosarcoma has been shown to also aberrantly express tissue factor, with descriptive studies suggesting similar effects on metastatic behavior. We hypothesized that tissue factor expressed on the surface of disseminated osteosarcoma cells activates procoagulant pathways and PAR signaling, and that both facilitate pulmonary metastasis. To test this, we first established and quantified expression of tissue factor across six osteosarcoma cell lines using Western blot, flow cytometry, and confocal microscopy in both monoculture and in coculture with human bronchial epithelial cells. PAR1 and PAR2 were also discovered to be expressed in osteosarcoma cells. RNA sequencing performed suggests that both tissue factor and PAR1 expression in osteosarcoma increase over the metastatic period, as does FVII expression by alveolar macrophages; this implies that tissue factor activation during metastasis is dynamic and dependent on host interactions. Ongoing work includes functional assays of osteosarcoma tissue factor and PARs activity as well as preclinical testing of tissue factor inhibition in a murine xenograft model of osteosarcoma. To define how tissue factor propagates metastasis, we are separately inhibiting its coagulant and cell signaling pathways after murine tail vein injection using apixaban or pathway-specific antibodies. We will also use an inducible F3 knockout to alter tumor expression of tissue factor. If we find that manipulation of tissue factor expression changes metastatic behavior across cell lines, this will suggest that tissue factor plays a crucial, mechanistic role in osteosarcoma metastasis. Varying effectiveness of coagulation or signaling pathway inhibition on metastasis will implicate tissue factor’s mechanism of metastatic propagation. Citation Format: Charles C. Treinen, Amy Gross, Maren Cam, Bryce Kerlin, Ryan Roberts. Identifying the role of tissue factor in osteosarcoma metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5456.

Abstract 1475: Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line

Abstract Prostate cancer (PCa) is the second most common cancer among males with an incidence rate of PCa is 288,300 new cases annually. Risk factors include age, genetics, and lifestyle, but the underlying stimuli remain largely uncertain. One potential emerging cause of PCa is exposure to metal carcinogens as cadmium (Cd) which is now classified as a group I human carcinogen. Chronic exposure to Cd on epithelial cells has been extensively shown to induce carcinogenesis, making it a likely contributor to PCa development. In this study, we expand this information by examining how chronic Cd exposure affects prostate-derived myofibroblast (WPMY-1) cells transforming into a cancer-associated fibroblast like phenotype. To study the transformation, WPMY-1 cells were treated with 500nM of Cd for a period of 11 months establishing the WPMY-1 Cd model. Fibroblasts are a crucial tumor microenvironment (TME) component and potentially contribute to PCa progression when transformed and activated into cancer-associated fibroblasts (CAFs). To begin, we used an MTT assay to measure the effect of Cd toxicity on normal prostate myofibroblasts versus WPMY-1 Cd. The data showed that 10µM Cd solution was toxic to WPMY-1 cells, but WPMY-Cd cells depicted high cell viability at 50 µM dose, conferring to chronic Cd exposure improved resiliency to Cd toxicity. We analyzed the expression of CAF-specific markers in WPMY-Cd cells, such as vimentin, FAP, α-SMA, and PDGFR-β. WPMY-Cd cells showed a distinct upregulation for these markers when compared to untreated WPMY-1 cells, suggesting that chronic exposure to Cd promotes CAF-like phenotypic transformation in WPMY-Cd. To further ascertain the cancer-promoting properties of WPMY-Cd, an in vitro 3-D spheroid assay was performed by growing the cells on a Matrigel scaffold. The WPMY-Cd cells formed many more tumor-like spheroid structures than WPMY-1 and exhibited sprouting. The sprouting spheroids displayed lamellipodium and filopodium-like structures, demonstrating the initiation of migratory phenotype in WPMY-Cd. Additionally, previous research suggests that CAFs may play a role in PCa progression by promoting tumor growth via the secretion of cytokines and growth factors. We tested this theory by exposing Cd-treated epithelial (BPH-Cd) cells to conditioned media containing secretions from CAFs and WPMY-Cd. WPMY-1 Cd conditioned media in conferring cell migration on epithelial BPH-Cd cells was confirmed by a wound healing assay wherein BPH-Cd cells treated with WPMY-1 Cd conditioned media led to complete wound closure in 24 hours as compared to WPMY-1 and CAF conditioned media. Overall, there is considerable evidence that Cd exposure in fibroblasts may play a crucial role in PCa disease progression and is a promising area for researching PCa treatment strategies against a crucial component of the tumor microenvironment. Citation Format: Sakshi V. Bansod, Gnanasekar Munarithinam. Investigating the carcinogenic effects and the mechanism of cadmium exposure on WPMY-1 prostate myofibroblast cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1475.

Abstract 5438: Circulating tumor cells exhibit a tumor stemness defense phenotype in Head and Neck cancer subjects undergoing cisplatin based chemotherapy

Abstract Introduction: We have proposed that tumor has its own defense mechanism similar to an organism having its own defense at the population level. We also proposed that the center of this tumor defense is the hypoxic niche of cancer stem cells (CSCs). Our lab has previously characterized the hypoxic niche of CSCs, and demonstrated that these CSCs reprogram to a highly inflammatory, aggressive and embryonic stemness phenotype during oxidative stress (1). These reprogrammed CSCs, the tumor stemness defense (TSD) phenotype is transient, and exert altruistic defense against bacterial invasion in SCC-25 cell line (2). Further, VEGF/VEGFR1 autocrine signaling was found in cisplatin induced TSD phenotype (4) and Myc/Hif2alpha stemness pathway was found in TSD phenotype of a leukemia stem cell model (3). We hypothesize that the platinum-induced stress may also activate TSD phenotype in the dormant CSC fraction of head and neck squamous cell carcinoma (HNSCC), which may mobilize to the distant tissue via circulation as circulating tumor cells (CTCs). Methods: To test the hypothesis, we isolated EpCAM+/ABCG2+ CTCs (blood, n=14) and EpCAM+/ABCG2+ CSCs (tumor tissue, n=6/14) of HNSCC subjects under cisplatin treatment. CTCs were collected from peripheral blood mononuclear cells (PBMNC) by the immunomagnetic sorting of EpCAM+ cells, and then cells were expanded in a defined serum free media that we used to maintain the hypoxic CSCs (1). We evaluated the EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs for TSD phenotype by performing qPCR gene expression, Boyden chamber assay of invasion, clonogenic assay and serial transplantation assay in NOD/SCID mice. Data was compared with the EpCAM+/ABCG2- cell population. We also developed a pre-clinical model of cisplatin-induced reprogramming of HNSCC derived CSCs to understand the molecular mechanisms. Results: In 8/14 patient derived EpCAM+/ABCG2+ CTCs and EpCAM+/ABCG2+ CSCs showed TSD phenotype associated gene expression, high migration/invasion, self sufficiency (ability to form spheroid when grown in serum free media without growth factors), enhanced secretion of protumorigenic growth factors; VEGF, SDF-1, PIGF, and HMGB1. These CTCs and CSCs have shown tumor formation with significant CSC frequency, when injected to NOD/SCID mice. Finally, using a SCC-25 cell line derived pre-clinical model, we demonstrated that cisplatin therapy reprogram CSCs to TSD phenotype. Conclusion: Our work indicates that CTCs with TSD phenotype may indicate the interaction between cisplatin and CSC niche defense. Using the pre-clinical model, the TSD phenotype based CSC niche defense mechanism may be further explored to develop markers for therapy response, metastasis or recurrence in HNSCC.

Neuroinflammation-induced neurodegeneration and associated microglia activation in Parkinson’s disease: a novel neurotherapeutic avenue

Parkinson’s disease (PD) is classified as one type of neurodegenerative disorder. Movement disorder, which includes resting tremors and slowness of movement, is a common clinical symptom in PD patients. Neuroinflammation is one of the most important processes involved in the pathogenesis of PD. An inflammatory response in the brain can induce neuronal cell death. Microglia, a type of immune cell, plays a crucial role in neuroinflammation. In this review, we discussed the information on microglia-activated neuroinflammation, its relationship with PD, and therapeutic approaches for neuroinflammation in PD. Under normal conditions, microglia in their inactive state (M0) act as surveillance agents in the brain to investigate potential invasions. They regulate neuron production, remodel synapses, and secrete growth factors to protect the neurons. Under pathological conditions, the M0 transforms into active phenotypes, dividing into pro-inflammatory (M1) and anti-inflammatory (M2) microglia. The M1 and M2 microglia exhibit opposite functions, where M1 microglia promote pro-inflammatory responses, and M2 microglia promote anti-inflammatory responses. This dichotomy of functions is essential for maintaining a healthy level of inflammation in the brain. Presently, multiple therapeutic strategies are available for PD, encompassing anti-inflammatory drugs, neuroprotective compounds, antioxidants, nanoparticles targeting neuroinflammation, stem cell interventions, lifestyle adjustments, and microglia-focused treatments. These treatments improve patients' movement, allowing them to have lifestyles like others, consequently benefiting their mental and emotional well-being. Preventing microglia from polarising into the M1 phenotype and promoting their polarisation into the M2 phenotype could be a challenging and promising approach for treating PD.

Human Fibroblast-Derived Matrix Hydrogel Accelerates Regenerative Wound Remodeling Through the Interactions with Macrophages.

Herein, a novel extracellular matrix (ECM) hydrogel is proposed fabricated solely from decellularized, human fibroblast-derived matrix (FDM) toward advanced wound healing. This FDM-gel is physically very stable and viscoelastic, while preserving the natural ECM diversity and various bioactive factors. Subcutaneously transplanted FDM-gel provided a permissive environment for innate immune cells infiltration. Compared to collagen hydrogel, excellent wound healing indications of FDM-gel treated in the full-thickness wounds are noticed, particularly hair follicle formation via highly upregulated β-catenin. Sequential analysis of the regenerated wound tissues disclosed that FDM-gel significantly alleviated pro-inflammatory cytokine and promoted M2-like macrophages, along with significantly elevated vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) level. A mechanistic study demonstrated that macrophages-FDM interactions through cell surface integrins α5β1 and α1β1 resulted in significant production of VEGF and bFGF, increased Akt phosphorylation, and upregulated matrix metalloproteinase-9 activity. Interestingly, blocking such interactions using specific inhibitors (ATN161 for α5β1 and obtustatin for α1β1) negatively affected those pro-healing growth factors secretion. Macrophages depletion animal model significantly attenuated the healing effect of FDM-gel. This study demonstrates that the FDM-gel is an excellent immunomodulatory material that is permissive for host cells infiltration, resorbable with time, and interactive with macrophages, where it thus enables regenerative matrix remodeling toward a complete wound healing.

Hormetic effects of a cannabinoid system component, 2-arachidonoyl glycerol, on cell viability and expression profile of growth factors in cultured mouse Sertoli cells: Friend or foe of male fertility?

The generally undesired effects of exocannabinoids on male reproduction include alterations in testicular cell proliferation and function, as well as apoptosis induction. However, this paradigm has been challenged by the ability of endocannabinoids to regulate reproductive function. The present study addresses these paradoxical facts by investigating the effects of the endocannabinoid 2-arachidonoyl glycerol (2-AG) on mouse Sertoli cells’ survival and apoptosis, with a mechanistic insight into Sertoli cell-based growth factors’ production. The Mus musculus Sertoli cell line (TM4) was exposed to different concentrations of 2-AG, and cell viability was evaluated using MTT assay. Growth factors’ gene and protein expressions were analyzed through RT-PCR and western blotting. 2-AG concentration dependently increased TM4 viability, with a slight increase starting at 0.0001 µM, a peak of 190% of the control level at 1 µM, and a decrease at 3 µM. Moreover, 2-AG paradoxically altered mRNA expression of caspase-3 and growth factors. Caspase-3 mRNA expression was down-regulated, and growth factors mRNA and protein expression were up-regulated when using a low concentration of 2-AG (1 μM). Opposite effects were observed by a higher concentration of 2-AG (3 μM). These paradoxical effects of 2-AG can be explained through the concept of hormesis. The results indicate the pivotal role of 2-AG in mediating Sertoli cell viability and apoptosis, at least in part, through altering growth factors secretion. Furthermore, they suggest the involvement of endocannabinoids in Sertoli cell-based physiological and pathological conditions and reflect the ability of abnormally elevated 2-AG to mimic the actions of exocannabinoids in reproductive dysfunction.

Self-Assembled DNA Composite-Engineered Mesenchymal Stem Cells for Improved Skin-Wound Repair.

The direct use of mesenchymal stem cells (MSCs) as therapeutics for skin injuries is a promising approach, yet it still faces several obstacles, including limited adhesion, retention, and engraftment of stem cells in the wound area, as well as impaired regenerative and healing functions. Here, DNA-based self-assembled composites are reported that can aid the adhesion of MSCs in skin wounds, enhance MSC viability, and accelerate wound closure and re-epithelialization. Rolling-circle amplification (RCA)-derived DNA flowers, equipped with multiple copies of cyclic Arg-Gly-Asp (cRGD) peptides and anti-von Willebrand factor (vWF) aptamers, act as robust scavengers of reactive oxygen species (ROS) and enable synergistic recognition and adhesion to stem cells and damaged vascular endothelial cells. These DNA structure-aided stem cells are retained at localized wound sites, maintain repair function, and promote angiogenesis and growth factor secretion. In both normal and diabetes-prone db/db mice models with excisional skin injuries, facile topical administration of DNA flower-MSCs elicits rapid blood vessel formation and enhances the sealing of the wound edges in a single dose. DNA composite-engineered stem cells warrant further exploration as a new strategy for the treatment of skin and tissue damage.

Combined transplantation of hiPSC-NSC and hMSC ameliorated neuroinflammation and promoted neuroregeneration in acute spinal cord injury

BackgroundSpinal cord injury (SCI) is a serious clinical condition that has pathological changes such as increased neuroinflammation and nerve tissue damage, which eventually manifests as fibrosis of the injured segment and the development of a spinal cord cavity leading to loss of function. Cell-based therapy, such as mesenchymal stem cells (MSCs) and neural stem cells (NSCs) are promising treatment strategies for spinal cord injury via immunological regulation and neural replacement respectively. However, therapeutic efficacy is rare reported on combined transplantation of MSC and NSC in acute mice spinal cord injury even the potential reinforcement might be foreseen. Therefore, this study was conducted to investigate the safety and efficacy of co-transplanting of MSC and NSC sheets into an SCI mice model on the locomotor function and pathological changes of injured spinal cord.MethodsTo evaluate the therapeutic effects of combination cells, acute SCI mice model were established and combined transplantation of hiPSC-NSCs and hMSCs into the lesion site immediately after the injury. Basso mouse scale was used to perform the open-field tests of hind limb motor function at days post-operation (dpo) 1, 3, 5, and 7 after SCI and every week after surgery. Spinal cord and serum samples were collected at dpo 7, 14, and 28 to detect inflammatory and neurotrophic factors. Hematoxylin–eosin (H&E) staining, masson staining and transmission electron microscopy were used to evaluate the morphological changes, fibrosis area and ultrastructure of the spinal cord.ResultM&N transplantation reduced fibrosis formation and the inflammation level while promoting the secretion of nerve growth factor and brain-derived neurotrophic factor. We observed significant reduction in damaged tissue and cavity area, with dramatic improvement in the M&N group. Compared with the Con group, the M&N group exhibited significantly improved behaviors, particularly limb coordination.ConclusionCombined transplantation of hiPSC-NSC and hMSC could significantly ameliorate neuroinflammation, promote neuroregeneration, and decrease spinal fibrosis degree in safe and effective pattern, which would be indicated as a novel potential cell treatment option.Graphical abstract

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

Abstract A084: AMH paracrine signaling between cancer and mesothelial cells modulates the ovarian tumor immune microenvironment

(058) Evaluation of Pathway Expression Following Down-Regulation of NELL1, a Potential Gene of Interest in Peyronie’s Disease

Abstract Introduction Peyronie’s Disease (PD) is a superficial fibrosing disorder characterized by inelastic fibrous plaques within the tunica albuginea. We previously performed high-resolution array comparative genomic hybridization analysis of genomic DNA from men with both PD and Dupuytren’s Disease (DD), yielding candidate genes, including NELL1, with potential involvement in these conditions. NELL1 is a secreted growth factor that enhances osteoblastic differentiation and chondrogenesis and may contribute to calcification and inflammation in fibrotic plaques via its interaction with bone morphogenetic protein (BMP) within the TGF-β signaling pathway. Here, we report on pathway analysis following up- and down-regulation of NELL1 using CRISPR modification in an in vitro setting. Objective This study aimed to investigate the influence of NELL1 modification on downstream gene regulation, focusing on pathways that were significantly regulated following NELL1 down-regulation. Methods For down-regulation of NELL1, gRNAs were delivered to HEK293 cells with a nuclease deficient (d) dCas9-KRAB system adapted from Streptococcus pyogenes. Gene regulation was assessed using qRT-PCR. Results For down-regulation of NELL1, PCR analysis confirmed a 95% reduction in expression. Non-target vectors did not change NELL1 expression levels. RNA sequencing results found over 5,000 differentially expressed genes between, with significant changes in multiple pathways, including the HIF-1α pathway, which promotes keloid development by activating the TGF-β/Smad pathways. Additionally, we identified pathways related to ECM-receptor interaction, inflammation, apoptosis, and cell cycle regulation that are mediated by TGF-β/Smad signaling, which plays a crucial role in fibrosis. Our analysis also demonstrated upregulation of the PI3K-Akt pathway, which is involved in regulating type I collagen expression in response to TGF-β1. These results highlight the significance of NELL1 in fibrotic regulation and suggest its potential involvement in the development of PD. Conclusions We have created guides for and successfully down-regulated the expression of NELL1, a gene of interest in Peyronie’s disease. We have also demonstrated, through RNA sequencing, that changes in NELL1 lead to large changes in gene and pathway expression. The identified modified pathways, including HIF-1α-mediated, TGF-β/Smad and PI3K-Akt, provide valuable insights into the potential molecular mechanisms underlying NELL1's role in regulation of fibrosis, and further investigation of these pathways may reveal potential therapeutic targets for fibrosis-related disorders. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Contraline, Techfields Inc, Alto Neurosciences, Curebase.

New Insights into Endothelial Dysfunction in Cardiometabolic Diseases: Potential Mechanisms and Clinical Implications.

The endothelium is a monocellular layer covering the inner surface of blood vessels. It maintains vascular homeostasis regulating vascular tone and permeability and exerts anti-inflammatory, antioxidant, anti-proliferative, and anti-thrombotic functions. When the endothelium is exposed to detrimental stimuli including hyperglycemia, hyperlipidemia, and neurohormonal imbalance, different biological pathways are activated leading to oxidative stress, endothelial dysfunction, increased secretion of adipokines, cytokines, endothelin-1, and fibroblast growth factor, and reduced nitric oxide production, leading eventually to a loss of integrity. Endothelial dysfunction has emerged as a hallmark of dysmetabolic vascular impairment and contributes to detrimental effects on cardiac metabolism and diastolic dysfunction, and to the development of cardiovascular diseases including heart failure. Different biomarkers of endothelial dysfunction have been proposed to predict cardiovascular diseases in order to identify microvascular and macrovascular damage and the development of atherosclerosis, particularly in metabolic disorders. Endothelial dysfunction also plays an important role in the development of severe COVID-19 and cardiovascular complications in dysmetabolic patients after SARS-CoV-2 infection. In this review, we will discuss the biological mechanisms involved in endothelial dysregulation in the context of cardiometabolic diseases as well as the available and promising biomarkers of endothelial dysfunction in clinical practice.

From Short Circuit to Completed Circuit: Conductive Hydrogel Facilitating Oral Wound Healing.

The primary challenges posed by oral mucosal diseases are their high incidence and the difficulty in managing symptoms. Inspired by the ability of bioelectricity to activate cells, accelerate metabolism, and enhance immunity, a conductive polyacrylamide/sodium alginate crosslinked hydrogel composite containing reduced graphene oxide (PAA-SA@rGO) is developed. This composite possesses antibacterial, anti-inflammatory, and antioxidant properties, serving as a bridge to turn the "short circuit" of the injured site into a "completed circuit," thereby prompting fibroblasts in proximity to the wound site to secrete growth factors and expedite tissue regeneration. Simultaneously, the PAA-SA@rGO hydrogel effectively seals wounds to form a barrier, exhibits antibacterial and anti-inflammatory properties, and prevents foreign bacterial invasion. As the electric field of the wound is rebuilt and repaired by the PAA-SA@rGO hydrogel, a 5 × 5 mm2 wound in the full-thickness buccal mucosa of rats can be expeditiously mended within mere 7 days. The theoretical calculations indicate that the PAA-SA@rGO hydrogel can aggregate and express SOX2, PITX1, and PITX2 at the wound site, which has a promoting effect on rapid wound healing. Importantly, this PAA-SA@rGO hydrogel has a fast curative effect and only needs to be applied for the first three days, which significantly improves patient satisfaction during treatment.

Tripterygium Glycosides in the Treatment of Asthma: Integrating Network Pharmacology, Molecular Docking and In Vitro Preliminary Pharmacological Evaluation

Objectives The mechanism of tripterygium glycosides (TG) in the treatment of asthma was explored by utilizing network pharmacology, molecular docking, and in vitro preliminary pharmacological tests. Methods An analysis of the bioactive ingredients of TG was conducted using the Traditional Chinese Medicine Systems Pharmacology Database database or predicted by FAFdrugs4, with relevant targets gathered from HERB, Swiss Target Prediction, STITCH, and SEA databases, and the targets for asthma were collected in Drugbank, DisGeNET, and GeneCards databases. Compound-target-pathway interactions and the function of putative targets were annotated by Cytoscape 3.6.0 and Database Visualization and Integrated Discovery, respectively. The affinity of the key ingredient and hub targets was assessed by molecular docking using AutoDock Vina software. Real-time quantitative polymerase chain reaction, cell-secreted cytokine assay, and ELISA were used to explore the effect of TG and its key ingredient triptolide (TP) for asthma. Results There were 12 bioactive ingredients in TG with 335 corresponding targets, 2672 asthma-associated targets, and 204 TG anti-asthma targets. TP and Tripterine were identified as the two key ingredients. RELA and TP53 exhibited the best topological index in protein-protein interaction network analysis and were regarded as hub targets. TG might play a role in biological processes such as lipopolysaccharide-mediated signaling pathway, regulation of nitric oxide biosynthetic process and mainly related to toll-like receptor, TNF and apoptosis signaling pathways. Molecular docking revealed that TP effectively bound to hub targets. In vitro experiments demonstrated that TG and TP reversed the RNA expression of airway remodeling marker CDH1 and FN. Moreover, TP reduced the secretion of vascular endothelial growth factor, implicating its role in airway remodeling. Conclusion TG might act on target genes RELA and TP53, utilizing the active ingredient TP, to regulate signaling pathways such as toll-like receptor, TNF, and apoptosis, thereby playing a role in the improving airway remodeling in asthma.

Insight into JNK Inhibition-based Strategy for the Treatment of Alzheimer's Disease

Background: Existing drugs for the treatment of Alzheimer's disease do not meet the basic requirements. Therefore, it is necessary to search for fundamentally new targets for the discovery of drugs for Alzheimer's disease (AD). When creating approaches to stimulate neurogenesis, such a search is promising to conduct as part of the development of a strategy for targeted regulation of intracellular signal transduction in regeneration-competent cells (RCCs). Moreover, the participation of c-Jun Nterminal kinases (JNK) in the regulation of the functions of the nervous tissue and neuroglial cells is known. Objective: The aim of the work was to study the effect of the JNK inhibitor (IQ-1S) on exploratory behavior and cognitive functions in aged mice (16-month-old male C57BL/6 mice as a model of AD) in combination with the dynamics of shifts in the RCCs functioning (nervous tissue progenitors: neural stem cells (NSCs) and neuronal-committed progenitors (NCPs); neuroglial cells: astrocytes, oligodendrocytes, microglial cells). Methods: The studies were carried out on male C57BL/6 mice aged 16 months as model of Alzheimer's disease. We studied the effect of the JNK inhibitor on exploratory behavior, conditioned reflex activity, and on the functioning of RCCs in the subventricular zone of the cerebral hemispheres (SVZ). NCPs and neuroglial cells of different types were obtained by immunomagnetic separation. Results: We observed significant changes in exploratory behavior and impaired conditioned reflex activity in aged C57BL/6 mice. The administration of the JNK inhibitor led to a significant correction of age-related behavioral and memory disorders in aged mice. At the same time, against the background of JNK inhibition-based therapy, an increase in mitotic activity and the content of both NSCs and NCPs in the SVZ was noted. However, these shifts were more pronounced in committed precursors. The phenomenon of the inhibition of NSC specialization under the influence of a pharmacological agent was also revealed. In addition, the JNK inhibitor caused an increase in the secretion of neurotrophic growth factors by oligodendrocytes and microglial cells. Conclusion: The findings open up prospects for the development of JNK targeting-based approaches for the treatment of AD.