Fibroblasts and endothelial cells interplay drives hypertrophic scar formation: Insights from in vitro and in vivo models.

Hypertrophic scar formation is influenced by the intricate interplay between fibroblasts and endothelial cells. In this study, we investigated this relationship using in vitro and in vivo models. Clinical observations revealed distinct morphological changes and increased vascularity at pathological scar sites. Further analysis using OCTA, immunohistochemistry, and immunofluorescence confirmed the involvement of angiogenesis in scar formation. Our indirect co-culture systems demonstrated that endothelial cells enhance the proliferation and migration of fibroblasts through the secretion of cytokines including VEGF, PDGF, bFGF, and TGF-ß. Additionally, a suspended co-culture multicellular spheroid model revealed molecular-level changes associated with extracellular matrix remodeling, cellular behaviors, inflammatory response, and pro-angiogenic activity. Furthermore, KEGG pathway analysis identified the involvement of TGF-ß, IL-17, Wnt, Notch, PI3K-Akt, and MAPK pathways in regulating fibroblasts activity. These findings underscore the critical role of fibroblasts-endothelial cells crosstalk in scar formation and provide potential targets for therapeutic intervention. Understanding the molecular mechanisms underlying this interplay holds promise for the development of innovative approaches to treat tissue injuries and diseases.

Notch4 participates in mesenchymal stem cell-induced differentiation in 3D-printed matrix and is implicated in eccrine sweat gland morphogenesis.

Background: Eccrine sweat gland (SG) plays a crucial role in thermoregulation but exhibits very limited regenerative potential. Although SG lineage-restricted niches dominate SG morphogenesis and benefit SG regeneration, rebuilding niches in vivo is challenging for stem cell therapeutic applications. Hence, we attempted to screen and tune the critical niche-responding genes that dually respond to both biochemical and structural cues, which might be a promising strategy for SG regeneration. Methods: An artificial SG lineage-restricted niche consisting of mouse plantar dermis homogenates (i.e. biochemical cues) and 3D architecture (i.e. structural cues) was built in vitro by using an extrusion-based 3D bioprinting approach. Mouse bone marrow-derived mesenchymal stem cells (MSCs) were then differentiated into the induced SG cells in the artificial SG lineage-restricted niche. To decouple biochemical cues from structural cues, the transcriptional changes aroused by pure biochemical cues, pure structural cues and synergistic effects of both cues were analyzed pairwise, respectively. Notably, only niche-dual-responding genes that are differentially expressed in response to both biochemical and structural cues and participate in switching MSC fates towards SG lineage were screened out. Validations in vitro and in vivo were respectively conducted by inhibiting or activating the candidate niche-dual-responding gene(s) to explore the consequent effects on SG differentiation. Results: Notch4 is one of the niche-dual-responding genes that enhanced MSC stemness and promoted SG differentiation in 3D-printed matrix in vitro. Furthermore, inhibiting Notch4 specifically reduced keratin 19-positive epidermal stem cells and keratin 14-positive SG progenitor cells, thus further delaying embryonic SG morphogenesis in vivo. Conclusions: Notch4 not only participates in mouse MSC-induced SG differentiation in vitro but is also implicated in mouse eccrine SG morphogenesis in vivo.

Integrating zinc/silicon dual ions with 3D-printed GelMA hydrogel promotes in situ hair follicle regeneration.

The regeneration of hair follicles lost from injury or disease represents a major challenge in cutaneous regenerative medicine. In this study, we investigated the synergetic effects between zinc and silicon ions on dermal cells and screened the optimal concentration of ions for medical applications. We integrated zinc/silicon dual ions into gelatin methacryloyl (GelMA) to bioprint a scaffold and determined that its mechanical properties are suitable for biological treatment. Then, the scaffold was employed to treat mouse excisional model in order to promote in situ hair follicle regeneration. Our findings showed that GelMA-zinc/silicon-printed hydrogel can significantly activate hair follicle stem cells and enhance neovascularization. The beneficial effects of the scaffold were further confirmed by the growth of hairs in the center of wounds and the improvement in perfusion recovery. Taken together, the present study is the first to combine GelMA with zinc/silicon dual ions to bioprint in situ for treating excisional wound, and this approach may regulate hair follicle regeneration not only directly by impacting stem cells but also indirectly through promoting angiogenesis.

Clinical outcomes of a novel porcine small intestinal submucosa patch for full-thickness hand skin defects: a retrospective investigation.

OBJECTIVE: To investigate the clinical outcomes of a novel soft tissue repair patch (porcine small intestinal submucosa patch, SIS patch) in the treatment of full-thickness hand skin defects. METHODS: From January 2017 to July 2019, 80 patients with hand soft tissue defects, who met the inclusion criteria, were retrospectively reviewed and divided into two groups. After debridement, patients in group A were treated with the novel SIS patch to cover the wound, and patients in group B were treated with autologous skin graft. The dimensions of skin defect area and healing outcome were evaluated and recorded. Scar assessment was carried out using Scar Cosmesis Assessment and Rating Scale (SCAR scale) at the last follow-up postoperation, and the recovery of wound sensation was assessed at the same time using British Medical Research Council (BMRC) grading of sensorimotor recovery. All the data were collected and statistically analyzed. RESULTS: A total of 80 patients were enrolled in the study with 40 patients in each group. Four patients in group A and 5 patients in group B were excluded due to wound infection and lost to follow-up. There were 36 patients in group A and 35 patients in group B finally got follow-up postoperation with mean interval of 12.75 ± 5.61 months in group A and 14.11 ± 5.42 months in group B. The dimensions of skin defect area in group A ranged from 7.5 to 87.5 cm2 (mean 25.97 ± 18.66 cm2) and in group B ranged from 7.5 to 86.25 cm2 (mean 33.61 ± 19.27 cm2) which have no significant difference (P > 0.05). SCAR scale results of group A and group B were 10.98 ± 0.33 and 9.49 ± 0.35, respectively, and the difference was statistically significant (P < 0.05). BMRC grading results showed 6 cases of S4, 11 cases of S3+, 5 cases of S3, 6 cases of S2, 6 cases of S1 and 2 cases of S0 in group A, and 8 cases of S4, 10 cases of S3+, 7 cases of S3, 4 cases of S2, 5 cases of S1, and 1 case of S0 in group B, which had no significant difference between them (P > 0.05). CONCLUSIONS: The novel SIS patch is an applicable biological material in the treatment of hand skin defect, which could achieve a better cosmetic appearance of the newborn skin tissue.

Reciprocal interaction between vascular niche and sweat gland promotes sweat gland regeneration.

The incorporation of vasculature is known to be effective in tissue or organ functional regeneration. However, a vague understanding of the interaction between epidermal appendages and their vascular niches is a foremost obstacle to obtaining sweat gland (SG)-specific vasculature units. Here, we map their precise anatomical connections and report that the interplay between SG cells (SGCs) and the surrounding vascular niche is key for glandular development and homeostasis maintenance. To replicate this interplay in vitro, we used three-dimensional (3D) bioprinting to generate reproducible SGC spheroids from differentiated adipose-derived mesenchymal stem cells (ADSCs). With dermal microvascular endothelial cells (DMECs), sacrificial templates made from poly (ε-caprolactone) (PCL) were fabricated to pattern the vascular niche. This interplay model promoted physiologically relevant vascularized glandular morphogenesis in vitro and in vivo. We identified a reciprocal regulatory mechanism for promoting SGs regeneration via contact-independent cell communication and direct cell-cell interactions between SGs and the vasculature. We envision the successful use of our approach for vascularized organ regeneration in the near future.

Collagen triple helix repeat containing-1 promotes functional recovery of sweat glands by inducing adjacent microvascular network reconstruction in vivo.

Background: Sweat glands (SGs) have low regenerative potential after severe burns or trauma and their regeneration or functional recovery still faces many obstacles. In practice, restoring SG function requires not only the structural integrity of the gland itself, but also its neighboring tissues, especially blood vessels. Collagen triple helix repeat containing-1 (CTHRC1) was first identified in vascular repair, and increasing reports showed a close correlation between cutaneous appendage specification, patterning and regeneration. The purpose of the present study was to clarify the role of CTHRC1 in SGs and their adjacent microvessels and find therapeutic strategies to restore SG function. Methods: The SGs and their adjacent microvascular network of Cthrc1 -/- mice were first investigated using sweat test, laser Doppler imaging, tissue clearing technique and transcriptome analysis. The effects of CTHRC1 on dermal microvascular endothelial cells (DMECs) were further explored with cell proliferation, DiI-labeled acetylated low-density lipoprotein uptake, tube formation and intercellular junction establishment assays. The effects of CTHRC1 on SG function restoration were finally confirmed by replenishing the protein into the paws of Cthrc1 -/- mice. Results: CTHRC1 is a key regulator of SG function in mice. At the tissue level, Cthrc1 deletion resulted in the disorder and reduction of the microvascular network around SGs. At the molecular level, the knockout of Cthrc1 reduced the expression of vascular development genes and functional proteins in the dermal tissues. Furthermore, CTHRC1 administration considerably enhanced SG function by inducing adjacent vascular network reconstruction. Conclusions: CTHRC1 promotes the development, morphogenesis and function execution of SGs and their neighboring vasculature. Our study provides a novel target for the restoration or regeneration of SG function in vivo.

Untargeted serum metabolomics and tryptophan metabolism profiling in type 2 diabetic patients with diabetic glomerulopathy.

Diabetic glomerulopathy (DG) remains the prevalent microvascular complication and leading cause of shortened lifespan in type-2 diabetes mellitus (T2DM) despite improvement in hyperglycemia control. Considering the pivotal role of kidney in metabolism, using untargeted metabolomic techniques to globally delineate the serum metabolite profiles will help advance understanding pathogenetic underpinnings of renal biopsy-confirmed DG from the perspective of metabolism specifically. Fourteen pathologically diagnosed DG patients secondary to T2DM and 14 age- and gender-matched healthy controls (HCs) were recruited for study. We employed mass spectrometry-based untargeted metabolomic methods to reveal the metabolite profiles of serum samples collected from all included subjects. We identified a total of 334 and 397 metabolites in positive and negative ion mode respectively. One hundred and eighty-two important differential metabolites whose variable importance in projection (VIP) > 1 and p value <0.05 were selected and annotated to metabolic pathways. KEGG pathway enrichment analysis revealed tryptophan metabolism enriched most significantly. Among the tryptophan derivatives, L-tryptophan (L-Trp) and serotonin were relatively accumulated in DGs compared with HCs, while 5-hydroxyindoleacetic acid (5-HIAA) and indole-3-acetamide were depleted. Correlation analysis showed serotonin and L-Trp are negatively correlated with 24 h urine protein and glycosylated hemoglobin (Ghb). To exclude the interference of preexisting T2DM on DG exacerbation, we selected 5-HIAA and 3-(3-hydroxyphenyl) propionic acid (3-OHPPA) which are not correlated with Ghb and analyzed their correlation relationship with crucial renal indices. We found 3-OHPPA is positively correlated with urine total protein and creatinine ratio (T/Cr) and 24 h urine protein, 5-HIAA is positively correlated with serum creatinine and urea.

Dietary Adherence, Self-Regulatory Fatigue and Trait Self-Control Among Chinese Patients with Peritoneal Dialysis: A Cross-Sectional Study.

PURPOSE: Little is known about factors that predict dietary adherence among Chinese patients undergoing peritoneal dialysis. We investigated whether self-regulatory fatigue and trait self-control influence dietary adherence among Chinese patients with peritoneal dialysis. METHODS: A total of 192 Chinese patients undergoing peritoneal dialysis were recruited at two peritoneal dialysis centers. The dietary adherence, trait self-control and self-regulatory fatigue of these patients were assessed using self-administered questionnaires. Clinical data were extracted from the hospital medical records. The significance of several social demographic factors on dietary adherence was analyzed using One-way ANOVA was used to analyze, whereas the association between dietary adherence and self-regulatory fatigue as well as trait self-control were analyzed using Pearson correlation. The independence association between dietary adherence and other influencing factors was assessed using Multivariable linear regression analyses. RESULTS: We found that peritoneal dialysis patients only moderately adhere to dietary prescriptions. The top three least adherences were observed for salt intake (1.89±0.36), face of difficulty (2.86±0.26) and fluid restriction adherence intake (2.97±0.30). Multivariable linear regression analysis revealed that education level (ß=0.339, P<0.001), residence (ß=-0.151, P=0.015), self-regulatory fatigue (ß= -0.648, P<0.001), and trait self-control (ß=0.118, P=0.022) were independent predictors of dietary adherence. CONCLUSION: Education level and residence strongly influence dietary adherence among Chinese peritoneal dialysis patients. On the other hand, Self-regulatory fatigue and trait self-control are independent predictors of dietary adherence among peritoneal dialysis patients. These findings can guide the enhancement of dietary adherence of peritoneal dialysis patients.

Nociceptor-localized cGMP-dependent protein kinase I is a critical generator for central sensitization and neuropathic pain.

Patients with neuropathic pain often experience exaggerated pain and anxiety. Central sensitization has been linked with the maintenance of neuropathic pain and may become an autonomous pain generator. Conversely, emerging evidence accumulated that central sensitization is initiated and maintained by ongoing nociceptive primary afferent inputs. However, it remains elusive what mechanisms underlie this phenomenon and which peripheral candidate contributes to central sensitization that accounts for pain hypersensitivity and pain-related anxiety. Previous studies have implicated peripherally localized cGMP-dependent protein kinase I (PKG-I) in plasticity of nociceptors and spinal synaptic transmission as well as inflammatory hyperalgesia. However, whether peripheral PKG-I contributes to cortical plasticity and hence maintains nerve injury-induced pain hypersensitivity and anxiety is unknown. Here, we demonstrated significant upregulation of PKG-I in ipsilateral L3 dorsal root ganglia (DRG), no change in L4 DRG, and downregulation in L5 DRG upon spared nerve injury. Genetic ablation of PKG-I specifically in nociceptors or post-treatment with intervertebral foramen injection of PKG-I antagonist, KT5823, attenuated the development and maintenance of spared nerve injury-induced bilateral pain hypersensitivity and anxiety. Mechanistic analysis revealed that activation of PKG-I in nociceptors is responsible for synaptic potentiation in the anterior cingulate cortex upon peripheral neuropathy through presynaptic mechanisms involving brain-derived neurotropic factor signaling. Our results revealed that PKG-I expressed in nociceptors is a key determinant for cingulate synaptic plasticity after nerve injury, which contributes to the maintenance of pain hypersensitivity and anxiety. Thereby, this study presents a strong basis for opening up a novel therapeutic target, PKG-I, in nociceptors for treatment of comorbidity of neuropathic pain and anxiety with least side effects.

The Alteration in Composition and Function of Gut Microbiome in Patients with Type 2 Diabetes.

BACKGROUND: Diabetes mellitus (DM) has become one of the most common chronic metabolic diseases worldwide. Due to the increasing prevalence and various complications, diabetes brings about a huge financial burden to DM patients. Nowadays, more and more studies reveal the relationship between diseases and gut microbial community. We aimed to explore the alteration in composition and function of the gut microbiome in T2DM patients. METHODS: A total of 137 patients with diabetes and 179 age- and gender-matched healthy controls selected from the healthy people sample center in the First Affiliated Hospital of Zhengzhou University were divided into the DM group and the Con group, respectively. We collected their venous blood for laboratory tests and stool samples for 16S rRNA sequencing. The comparison between the two groups including both composition and function of the gut microbiome is presented. RESULTS: We found that the α-diversity of bacterial taxa in the DM group had an evident decrease compared to that in the Con group. At the phylum level, the DM group had an obvious decrease of Bacteroidetes and a marked increase of Proteobacteria, Actinobacteria, and Verrucomicrobia. At the genus level, Bacteroides and Prevotella decreased the most while Escherichia-Shigella, Lachnospiraceae_incertae_sedis, Subdoligranulum, Enterococcus, and Klebsiella had different degrees of expansion in the DM group. The ROC based on 246 optimum OTUs had very high test efficiency with an AUC of 92.25% in the training set and 90.48% in the test set. As for prediction of metabolic function, the gut microbiome of DM patients was predicted to be more active in environmental information processing and human diseases but less in metabolism. CONCLUSION: We observed alteration of composition and function of the gut microbiome in the DM group. These changes may provide a new treatment strategy for DM patients and new research targets.

The gut microbiome in differential diagnosis of diabetic kidney disease and membranous nephropathy.

BACKGROUND: Diabetic kidney disease (DKD) and membranous nephropathy (MN) are the two major causes of end-stage renal disease (ESRD). Increasing evidence has shown that intestinal dysbiosis is associated with many diseases. The aim of this study was to explore the composition of the gut microbiome in DKD and MN patients. METHODS: 16S rRNA gene sequencing was performed on 271 fecal samples (DKD = 129 and MN = 142), and taxonomic annotation of microbial composition and function was completed. RESULTS: We observed distinct microbial communities between the two groups, with MN samples exhibiting more severe dysbiosis than DKD samples. Relative increases in genera producing short-chain fatty acids (SCFAs) in DKD and a higher proportion of potential pathogens in MN were the main contributors to the microbiome alterations in the two groups. Five-fold cross-validation was performed on a random forest model, and four operational taxonomic unit (OTU)-based microbial markers were selected to distinguish DKD from MN. The results showed 92.42% accuracy in the training set and 94.52% accuracy in the testing set, indicating high potential for these microbiome-based markers in separating MN from DKD. Overexpression of several amino acid metabolic pathways, carbohydrate metabolism and lipid metabolism was found in DKD, while interconversion of pentose/glucoronate and membrane transport in relation to ABC transporters and the phosphotransferase system were increased in MN. CONCLUSION: The composition of the gut microbiome appears to differ considerably between patients with DKD and those with MN. Thus, microbiome-based markers could be used as an alternative tool to distinguish DKD and MN.

miR-15a-5p suppresses peritoneal fibrosis induced by peritoneal dialysis via targeting VEGF in rats.

AIM: When peritoneal fibrosis (PF) causes ultrafiltration failure in peritoneal dialysis (PD) patients, PD has to be discontinued. Currently, there is no effective way to relieve PF. In this study, we aimed to determine whether miR-15a-5p is involved in PF and to determine the underlying mechanism. METHODS: Six normal rats were used as the control group. A uremic rat model was constructed using 5/6 nephrectomy in a Sprague-Dawley model. The uremic rats were randomly divided into PD, lentivirus-transfected, negative control, VEGFR-inhibited and gavage control groups. Except for the control group, all uremia rats received continuous PD for 28 days. In the lentivirus-transfected group, the miR-15a-5p plasmid was injected into the peritoneal cavity to upregulate miR-15a-5p expression. Axitinib was used to block vascular endothelial growth factor receptor (VEGFR) in the peritoneum. The mRNA levels of miR-15a-5p and VEGF were detected by qRT-PCR and FISH. Protein levels of VEGF, E-cadherin, collagen IV, fibronectin and α-SMA were detected by western blot and immunohistochemistry. RESULTS: PD leads to peritoneal thickening and fibrosis. The expression level of miR-15a-5p decreased and that of VEGF increased in the PD group than in the controls. Additionally, E-cadherin was significantly reduced while collagen IV, fibronectin and α-SMA were obviously increased in the PD group compared to controls. FISH showed that VEGF might be the target gene of miR-15a-5p. Overexpression of miR-15a-5p or inhibition of VEGFR could reverse PF. CONCLUSION: miR-15a-5p may participate in the endothelial to mesenchymal transition of PF caused by PD through VEGF.