Insulin-like growth factor-2 mRNA-binding protein 2 facilitates post-ischemic angiogenesis by increasing the stability of fibroblast growth factor 2 mRNA and its protein expression.

Background: Post-ischemic angiogenesis is crucial for reestablishing blood flow in conditions such as peripheral artery disease (PAD). The role of insulin-like growth factor-2 mRNA-binding protein 2 (IGF2BP2) in post-transcriptional RNA metabolism and its involvement in post-ischemic angiogenesis remains unclear. Methods: Using a human GEO database and a hind-limb ischemia (HLI) mouse model, the predominant isoform IGF2BP2 in ischemic gastrocnemius tissue was identified. Adeno-associated virus with the Tie1 promoter induced IGF2BP2 overexpression in the HLI model, evaluating the expression of vascular structural proteins (CD31 and α-SMA) and blood flow recovery after HLI. In vitro experiments with human umbilical vein endothelial cells (HUVECs) demonstrated that lentivirus-mediated IGF2BP2 overexpression upregulates cell proliferation, migration, and tube formation. GeneCards, RNAct databases, and subsequent reverse transcription quantitative polymerase chain reaction (RT-qPCR) predicted IGF2BP2 interactions with fibroblast growth factor 2 (FGF2) mRNA, and actinomycin D treatment, binding site predictions and CLIP-seq data further confirmed this interaction. Furthermore, western blotting, enzyme-linked immunosorbent assay, and RNA immunoprecipitation followed by RT-qPCR were performed to validate IGF2BP2's interaction with FGF2 mRNA and to assess its role in stabilizing FGF2 mRNA, as well as its impact on FGF2 protein expression. Results: HLI reduced IGF2BP2 expression in the gastrocnemius tissue, which gradually increased during blood flow recovery. IGF2BP2 overexpression in HLI mice accelerated blood flow recovery and increased capillary and small artery densities. The overexpression of IGF2BP2 in HUVECs stimulated proliferation, migration, and tube formation by interacting with FGF2 mRNA to increase its stability. This interaction resulted in increased levels of FGF2 protein and secretion, ultimately promoting angiogenesis. Conclusions: IGF2BP2 contributes to blood flow restoration post-ischemia in vivo and promotes angiogenesis in HUVECs by enhancing FGF2 mRNA stability and FGF2 protein expression and secretion. These findings underscore IGF2BP2's therapeutic potential in ischemic conditions, such as PAD.

LPS induces RGS-1 to promote infectious intracranial aneurysm formation and rupture by accelerating smooth muscle cell phenotypic switching.

OBJECTIVE: Patients with infectious intracranial aneurysms (IIAs) have high mortality rates. Sepsis is an important condition that induces IIA. Smooth muscle cell (SMC) phenotypic switching may have a critical effect on sepsis-induced IIA, but its role remains unclear. Hence, we aimed to identify sepsis-induced target genes involved in SMC phenotypic switching and their underlying mechanisms. METHODS AND RESULTS: RNA sequencing and bioinformatics analyses of samples from patients with intracranial aneurysms and sepsis identified RGS-1 as a common differentially expressed gene (DEG) involved in SMC phenotypic switching. Experimental verification demonstrated that lipopolysaccharide (LPS), a critical molecule in sepsis, increased RGS-1 levels, promoted SMC phenotypic switching and proliferation, and upregulated the expression of matrix metalloproteinases and inflammatory factors. Furthermore, qRT-PCR and immunofluorescence experiments confirmed that RGS-1 knockdown under LPS stimulation inhibited SMC phenotypic switching, cell proliferation, and decreases in matrix metalloproteinases and inflammatory factors. Mechanistically, western blotting, bioinformatics analyses, and chip assays revealed that RGS-1 activates the JNK-P38 pathway to promote SMC phenotypic switching and is regulated by the transcription factor STAT1. CONCLUSION: LPS induces RGS-1 to promote IIA formation and rupture by accelerating SMC phenotypic switching.

Association between periprocedural myocardial injury and neointimal characteristics in patients with in-stent restenosis: an optical coherence tomography study.

Background: The relationship between neointimal characteristics of in-stent restenosis (ISR) and periprocedural myocardial injury (PMI) remains unclear. Therefore, this study aimed to investigate the relationship between PMI and neointimal characteristics of ISR by using optical coherence tomography (OCT). Methods: This was a retrospective study. We enrolled 140 patients diagnosed with ISR with normal baseline high-sensitivity troponin T (hs-cTnT) levels who underwent OCT and subsequent revascularization by means of drug-coated balloon (DCB) or drug-eluting stent (DES) between October 2018 and October 2022 in the Affiliated Hospital of Zunyi Medical University. Based on the 4th universal definition of myocardial infarction, patients whose hs-cTnT were increased five times above the upper reference limit (URL) after percutaneous coronary interventions (PCI) were deemed to PMI. The patients were subdivided into PMI (n=53) and non-PMI (n=87) groups. In the univariable analysis, variables in the baselines, angiography characteristics and OCT findings were analyzed with binary logistic regression. A P value of <0.2 was included in the multivariable model. Multivariable logistic regression analysis was used to identify the independent predictors of PMI. Results: The prevalence of intra-intimal microvessels in patients with PMI was higher than in those without PMI (58.5% vs. 32.2%, P=0.003). The ratio of intra-stent plaque rupture (PR) was also higher in patients with PMI (60.4% vs. 40.2%, P=0.021). Multivariable logistic regression analysis showed that intra-intimal microvessels [odds ratio (OR): 3.193, 95% confidence interval (CI): 1.280-7.966; P=0.013] and intra-stent PR (OR: 2.124, 95% CI: 1.153-4.732; P=0.035) were independently associated with PMI. Conclusions: Intra-intimal microvessels and intra-stent PR were independently associated with PMI. Accurate identification and recognition of intra-intimal microvessels and intra-stent PR may be helpful in preventing PMI.

Association of the monocytes to high-density lipoprotein cholesterol ratio with in-stent neoatherosclerosis and plaque vulnerability: An optical coherence tomography study.

BACKGROUND: Monocyte-to-high-density lipoprotein cholesterol ratio (MHR) is an independent predictor of atherosclerosis and in-stent restenosis (ISR). However, the association between MHR and the incidence of in-stent neoatherosclerosis (ISNA) remains to be validated. METHODS: This study included 216 patients with acute coronary syndrome who had 220 ISR lesions and had undergone optical coherence tomography (OCT). All eligible patients were divided into three groups according to their MHR tertile level. OCT characteristics were comparatively analyzed between groups of different MHR levels, and univariate and multivariate logistic regression analyses were constructed to assess correlations between MHR level and ISNA as well as in-stent thin-cap fibroatheroma (TCFA). A receiver operating characteristic curve was used to determine the optimal MHR thresholds for predicting ISNA and in-stent TCFA. RESULTS: The incidence of ISNA (70.3% vs. 61.1% vs. 20.3%, P < 0.001) and in-stent TCFA (40.5% vs. 31.9% vs. 6.8%, P < 0.001) was the highest in the third tertile, followed by the second and first tertiles, respectively. Multivariate analysis revealed that MHR was independently associated with ISNA (odds ratio [OR], 7.212; 95% confidence interval [CI], 1.287-40.416; P = 0.025) and in-stent TCFA (OR, 5.610; 95% CI, 1.743-18.051; P = 0.004) after adjusting for other clinical factors. The area under the curve was 0.745 (95% CI, 0.678-0.811; P < 0.001) for the prediction of ISNA and 0.718 (95% CI, 0.637-0.778; P < 0.001) for the prediction of in-stent TCFA. CONCLUSION: MHR levels are an independent risk factor for ISNA.

Machine learning for predicting intrahospital mortality in ST-elevation myocardial infarction patients with type 2 diabetes mellitus.

In an era of increasing need for precision medicine, machine learning has shown promise in making accurate acute myocardial infarction outcome predictions. The accurate assessment of high-risk patients is a crucial component of clinical practice. Type 2 diabetes mellitus (T2DM) complicates ST-segment elevation myocardial infarction (STEMI), and currently, there is no practical method for predicting or monitoring patient prognosis. The objective of the study was to compare the ability of machine learning models to predict in-hospital mortality among STEMI patients with T2DM. We compared six machine learning models, including random forest (RF), CatBoost classifier (CatBoost), naive Bayes (NB), extreme gradient boosting (XGBoost), gradient boosting classifier (GBC), and logistic regression (LR), with the Global Registry of Acute Coronary Events (GRACE) risk score. From January 2016 to January 2020, we enrolled patients aged > 18 years with STEMI and T2DM at the Affiliated Hospital of Zunyi Medical University. Overall, 438 patients were enrolled in the study [median age, 62 years; male, 312 (71%); death, 42 (9.5%]). All patients underwent emergency percutaneous coronary intervention (PCI), and 306 patients with STEMI who underwent PCI were enrolled as the training cohort. Six machine learning algorithms were used to establish the best-fit risk model. An additional 132 patients were recruited as a test cohort to validate the model. The ability of the GRACE score and six algorithm models to predict in-hospital mortality was evaluated. Seven models, including the GRACE risk model, showed an area under the curve (AUC) between 0.73 and 0.91. Among all models, with an accuracy of 0.93, AUC of 0.92, precision of 0.79, and F1 value of 0.57, the CatBoost model demonstrated the best predictive performance. A machine learning algorithm, such as the CatBoost model, may prove clinically beneficial and assist clinicians in tailoring precise management of STEMI patients and predicting in-hospital mortality complicated by T2DM.

Association of LDL-C level with neoatherosclerosis and plaque vulnerability in patients with late restenosis: an optical coherence tomography study.

Neoatherosclerosis (NA) is a significant contributor to late stent failure; however, predictors of late in-stent restenosis (ISR) with NA have not been systematically reported. This study aimed to identify predictors of NA incidence and plaque vulnerability in patients with late ISR and the role of low-density lipoprotein cholesterol (LDL-C) levels in this process. A total of 216 patients with 216 lesions who underwent optical coherence tomography (OCT) before interventional procedure for late drug-eluting stent ISR were enrolled and divided into NA and non-NA groups based on OCT findings. Results showed that higher LDL-C levels were associated with NA, thin-cap fibroatheroma (TCFA), intimal disruption, plaque erosion, and thrombosis. Multivariate regression analysis revealed that the LDL-C level was an independent risk factor for NA and TCFA. The LDL-C levels exhibited a significant predictive value for NA and TCFA, surpassing other factors such as stent age and other lipid types. In conclusion, a high LDL-C level is an independent predictor of NA incidence and plaque vulnerability in patients with late ISR.

Construction of calcitonin gene-related peptide-modified mesenchymal stem cells and analysis of their effects on the migration and proliferation of vascular smooth muscle cells.

Lentiviral expression vectors for calcitonin gene-related peptide (CGRP) were used to transfect rat bone marrow mesenchymal stem cells (MSCs). After assessing the biological characteristics of proliferation and aging in MSCs transfected with CGRP, we observed the effects of the CGRP-modified rat MSCs on the migration and proliferation of rat vascular smooth muscle cells (VSMCs) in vitro. Rat MSCs were isolated, cultured in vitro, and identified by flow cytometry. A CGRP recombinant lentivirus was transfected into MSCs. The transfection efficiency was determined by fluorescence microscopy and flow cytometry, and CGRP in MSCs was detected by real-time quantitative PCR, ELISA, and immunofluorescence. The proliferation and senescence of CGRP-modified MSCs were evaluated by MTT assay and beta-galactosidase staining. VSMCs were isolated, cultured in vitro, and identified by immunofluorescence. CGRP-modified MSCs and VSMCs were cocultured in a Transwell system. The proliferation and migration of VSMCs were evaluated by scratch testing and the MTT method. Rat bone marrow MSCs showed a spindle-shaped morphology, adherent growth in vitro, positive CD29 and CD90 expression, and negative CD45 expression. CGRP was stably expressed in MSCs after 48 h of recombinant lentivirus transfection. CGRP mRNA and protein secretion in CGRP recombinant lentivirus-transfected MSCs were higher than that in control MSCs. Immunofluorescence showed that CGRP protein could be expressed in CGRP-modified MSCs. The proliferation ability and senescence rates did not differ between lentivirus-transfected MSCs and untransfected MSCs. Rat VSMCs expressed α-SMA protein and exhibited a spindle-shaped morphology and adherent growth in vitro. In Transwell coculture experiments, scratch testing of VSMCs showed that CGRP-modified MSCs could reduce VSMC proliferation and migration. The CGRP gene can be stably expressed in MSCs after CGRP recombinant lentivirus transfection. CGRP recombinant lentivirus transfection has little effect on the proliferation or senescence of MSCs, and CGRP-modified MSCs can inhibit the proliferation and migration of VSMCs. These results lay a foundation for research on the use of CGRP gene-engineered MSCs in restenosis therapy.

[Role of CGRP modified mesenchymal stem cells in restenosis in rat model with carotid angioplasty].

OBJECTIVE: To explore the role of calcitonin gene-related peptide (CGRP) in the proliferation of vascular smooth muscle cells (VSMCs) and restenosis. METHODS: The high-expression CGRP lentivirus [Lenti-green fluorescent protein (GFP)-CGRP] was constructed. And mesenchymal stem cells (MSCs) were transfected with Lenti-GFP-CGRP, Lenti-GFP and phosphate buffer saline (PBS). Reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to determine the CGRP gene and protein expression level of smooth muscle cells in each group respectively. Then MSCs were co-cultured with VSMCs. Experimental groups were MSCs(CGRP+/+)+VSMCs, MSCs(CGRP-/-)+VSMCs and MSCs(PBS)+VSMCs groups. The method of methyl thiazolyl tetrazolium (MTT) was employed to detect the proliferation of smooth muscle cells. Sacculus damaged atherosclerotic carotid was prepared according to the previous study. MSCs were transfected with Lv-CGRP-EGFP and Lv-CGRP and then transplanted into rat model. At Day 7 post-transplantation, injured carotid artery was harvested to detect the C expression of GRP by Western blot and the expression of CD31 by immunofluorescence. At Day 28 post-transplantation, injured carotid artery was harvested to assess organization morphology by hematoxylin and eosin stain and detect the expression of proliferating cell nuclear antigen (PCNA) by immunohistochemical stain. RESULTS: As compared with control and Lenti-GFP groups, the expressions of CGRP protein and CGRP mRNA increased at 72 h after transfecting with Lenti-GFP-CGRP (P < 0.05). After 72-hour co-culturing with VSMCs, the proliferation ability of VSMCs was the lowest in MSCs(CGRP+/+)+VSMCs group versus other three groups (P < 0.05). At Day 7 post-transplantation, as compared with control and Lenti-GFP groups, the expression of CGRP proteins increased significantly in MSCs-CGRP group (P < 0.05). A continuous expression of CD31 was found in damaged carotid intima in MSCs-CGRP group, but not in control group. At Day 28 post-transplantation, the area of intimal hyperplasia was smaller in MSCs-CGRP group than that in control and Lenti-GFP groups. Also the expression of PCNA decreased more in MSCs-CGRP group than control and Lenti-GFP groups (P < 0.05). CONCLUSIONS: CGRP protein and CGRP mRNA are expressed after CGRP transfection. And CGRP can suppress the proliferation of VSMCs and reduce intimal hyperplasia so as to facilitate a recovery of damaged endothelium.

[Impact and related mechanism of exogenous receptor activity modifying protein 1 on calcitonin gene-related peptide modified bone marrow mesenchymal stem cells on the migration of vascular smooth muscle cells in vitro].

OBJECTIVE: To investigate the impact of calcitonin gene-related peptide (CGRP) modified bone marrow mesenchymal stem cell (MSC) on the migration of vascular smooth muscle cell (VSMC) and related mechanisms. METHODS: The MSC and VSMC were isolated from rats and cultured, CGRP was transfected to MSC with the high expression lentivirus vector, VSMC was transfected with high expression lentivirus vector of receptor activity modifying protein 1 (RAMP1) and the silence expression lentivirus vector of RAMP1. Then MSC was co-cultured with VSMC. Experimental groups were as follows: (1) Ang II group (MSC + VSMC + Ang II); (2) MSC(CGRP+) group (MSC(CGRP+) + VSMC + Ang II); (3) MSC(CGRP+) RAMP1(-) group (MSC(CGRP+) + VSMC(RAMP1-) + Ang II); (4) MSC(CGRP+) RAMP1(+) group (MSC(CGRP+) + VSMC(RAMP1+) + Ang II); (5) RAMP1(+) group (MSC + VSMC(RAMP1+) + Ang II). Transwell assay was applied to detect the migration of smooth muscle cells, Western blot was applied to detect the protein expression of cells in various groups. RESULTS: VSMC migration number was significantly lower in MSC(CGRP+) group compared with Ang II group (50.8 ± 2.6 vs. 71.4 ± 2.3, P < 0.05), but higher than in MSC(CGRP+) RAMP1(+) group (50.8 ± 2.6 vs. 30.4 ± 3.0, P < 0.05). When RAMP1 expression reduced in VSMC, compared with MSC(CGRP+) RAMP1(+) group, VSMC migration increased in the MSC(CGRP+) RAMP1(-) group compared to MSC(CGRP+)RAMP1(+) (69.0 ± 5.6 vs. 30.4 ± 3.0, P < 0.05) and was similar to Ang II group (69.0 ± 5.6 vs. 71.4 ± 2.3, P > 0.05) and RAMP1(+) group (71.6 ± 3.4). According to the result of Western blot, P-P65 protein expression in MSC(CGRP+) group was lower than that in Ang II group (0.475 ± 0.022 vs.0.642 ± 0.035, P < 0.05). P-P65 protein expression in MSC(CGRP+)RAMP1(-) group was higher than that in MSC(CGRP+) RAMP1(+) group (0.670 ± 0.030 vs. 0.373 ± 0.041, P < 0.05), and there was no difference between MSC(CGRP+)RAMP1(-) group and Ang II group (P > 0.05). P-P65 protein expression was similar between RAMP1(+) group (0.643 ± 0.039) and Ang II group (P > 0.05). CONCLUSIONS: CGRP inhibits VSMC migration through RAMP1. NF-κB and RAMP1 play crucial role in the inhibiting effects of CGRP on VSMC migration. Thus, RAMP1-CGRP signaling inhibits VSMC migration through NF-κB signal pathways.

Molecular design of modified polyacrylamide for the salt tolerance.

In our work, three kinds of functional monomers were selected to modify polyacrylamide (PAM) or partially hydrolyzed polyacrylamide (HPAM) by molecular dynamics simulation so as to achieve the stronger salt-tolerance of modified HM-HPAM. The radius of gyration (R (g)), the hydrodynamic radius (R (H)), the effective length (L (ef)) and the intrinsic viscosity ([η]) for modified PAM or HPAM were studied in aqueous solutions with different ionic strength at 298 K. The results showed that modified HM-HPAM has a stronger salt tolerance and the salt tolerance increases gradually from HM-HPAM1 to HM-HPAM3 because the monomers with different steric hindrance would reduce the curliness of molecular chains and, consequently, improve the salt tolerance. So, introducing the steric hindrance monomer into polymer will increase the salt tolerance of the polymer and it is indicated that the simulated results agree with the experimental results very well. Furthermore, the radial distribution function (RDF) has been used to investigate the effect of NaCl on the hydration of the -COO- groups of the HM-HPAM from microscopic view.

Experimental and theoretical study of dilute polyacrylamide solutions: effect of salt concentration.

The structure and intrinsic viscosity of the partially hydrolyzed polyacrylamide (HAPM) and polyacrylamide (PAM) in aqueous solution were investigated by comparative studies of molecular dynamics simulation over a wide range of the NaCl concentration. The radius of gyration (R(g)), the hydrodynamic radius (R(h)) and the ratio of the radius gyration and the hydrodynamic radius (ρ) were calculated for the PAM or HPAM in solutions with different NaCl concentrations at 298 K. The conformational changes of the polymer chain in different aqueous solution were discussed according to the molecular shapes. It was found that the change of the R(h) or the R(g) can reflect the change in the [η]. And the changes in the structure of the polymer chain with different NaCl concentrations were discussed via the ρ which can predict [η] changes. The results showed that behavior of the polymer solution calculated from the simulation agreed with the experimental measurements. Furthermore, the radial distribution functions for the HPAM solutions were investigated, which verified the micro-mechanism for the change of the structure. The results of this research showed that the computational method used in this work has practical applicability.