Single-cell RNA sequencing in donor and end-stage heart failure patients identifies NLRP3 as a therapeutic target for arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Dilation may be the first right ventricular change and accelerates the progression of threatening ventricular tachyarrhythmias and heart failure for patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), but the treatment for right ventricular dilation remains limited. METHODS: Single-cell RNA sequencing (scRNA-seq) of blood and biventricular myocardium from 8 study participants was performed, including 6 end-stage heart failure patients with ARVC and 2 normal controls. ScRNA-seq data was then deeply analyzed, including cluster annotation, cellular proportion calculation, and characterization of cellular developmental trajectories and interactions. An integrative analysis of our single-cell data and published genome-wide association study-based data provided insights into the cell-specific contributions to the cardiac arrhythmia phenotype of ARVC. Desmoglein 2 (Dsg2)mut/mut mice were used as the ARVC model to verify the therapeutic effects of pharmacological intervention on identified cellular cluster. RESULTS: Right ventricle of ARVC was enriched of CCL3+ proinflammatory macrophages and TNMD+ fibroblasts. Fibroblasts were preferentially affected in ARVC and perturbations associated with ARVC overlap with those reside in genetic variants associated with cardiac arrhythmia. Proinflammatory macrophages strongly interact with fibroblast. Pharmacological inhibition of Nod-like receptor protein 3 (NLRP3), a transcriptional factor predominantly expressed by the CCL3+ proinflammatory macrophages and several other myeloid subclusters, could significantly alleviate right ventricular dilation and dysfunction in Dsg2mut/mut mice (an ARVC mouse model). CONCLUSIONS: This study provided a comprehensive analysis of the lineage-specific changes in the blood and myocardium from ARVC patients at a single-cell resolution. Pharmacological inhibition of NLRP3 could prevent right ventricular dilation and dysfunction of mice with ARVC.

Single-Cell RNA Sequencing of Coronary Perivascular Adipose Tissue From End-Stage Heart Failure Patients Identifies SPP1+ Macrophage Subpopulation as a Target for Alleviating Fibrosis.

BACKGROUND: Perivascular adipose tissue (PVAT) is vital for vascular homeostasis, and PVAT dysfunction is associated with increased atherosclerotic plaque burden. But the mechanisms underlining coronary PVAT dysfunction in coronary atherosclerosis remain elusive. METHODS: We performed single-cell RNA sequencing of the stromal vascular fraction of coronary PVAT from 3 groups of heart transplant recipients with end-stage heart failure, including 3 patients with nonobstructive coronary atherosclerosis, 3 patients with obstructive coronary artery atherosclerosis, and 4 nonatherosclerosis control subjects. Bioinformatics was used to annotate the cellular populations, depict the cellular developmental trajectories and interactions, and explore the differences among 3 groups of coronary PVAT at the cellular and molecular levels. Pathological staining, quantitative real-time polymerase chain reaction, and in vitro studies were performed to validate the key findings. RESULTS: Ten cell types were identified among 67 936 cells from human coronary PVAT. Several cellular subpopulations, including SPP1+ (secreted phosphoprotein 1) macrophages and profibrotic fibroadipogenic progenitor cells, were accumulated in PVAT surrounding atherosclerotic coronary arteries compared with nonatherosclerosis coronary arteries. The fibrosis percentage was increased in PVAT surrounding atherosclerotic coronary arteries, and it was positively associated with the grade of coronary artery stenosis. Cellular interaction analysis suggested OPN (osteopontin) secreted by SPP1+ macrophages interacted with CD44 (cluster of differentiation 44)/integrin on fibroadipogenic progenitor cells. Strikingly, correlation analyses uncovered that higher level of SPP1 in PVAT correlates with a more severe fibrosis degree and a higher coronary stenosis grade. In vitro studies showed that conditioned medium from atherosclerotic coronary PVAT promoted the migration and proliferation of fibroadipogenic progenitor cells, while such effect was prevented by blocking CD44 or integrin. CONCLUSIONS: SPP1+ macrophages accumulated in the PVAT surrounding atherosclerotic coronary arteries, and they promoted the migration and proliferation of fibroadipogenic progenitor cells via OPN-CD44/integrin interaction and thus aggravated the fibrosis of coronary PVAT, which was positively correlated to the coronary stenosis burden. Therefore, SPP1+ macrophages in coronary PVAT may participate in the progression of coronary atherosclerosis.

Wnt4-modified NSC transplantation promotes functional recovery after spinal cord injury.

Spinal cord injury (SCI) can lead to severe motor and sensory dysfunction, yet there are no effective therapies currently due to the failure of reconstructing the interruption of the neuroanatomical circuit. While neural stem cell (NSC) transplantation has been considered a potential strategy to repair the neural circuit after SCI, the efficacy of this strategy remains unproven. The main reason is that most of the transplanted NSC differentiates into astrocyte rather than neuron in the microenvironment of SCI. Our results demonstrated that Wnt4 significantly promotes the differentiation of NSC into neuron by activating both ß-catenin and MAPK/JNK pathways and suppressing the activation of Notch signaling, which is acknowledged as prevention of NSC differentiation into neuron, through downregulating NICD expression, translocating and preventing the combination of NICD and RbpJ in nucleus. In addition, Wnt4 rescues the negative effect of Jagged, the ligand of Notch signaling, to promote neuronal differentiation. Moreover, in vivo study, transplantation of Wnt4-modified NSC efficaciously repairs the injured spinal cord and recovers the motor function of hind limbs after SCI. This study sheds new light into mechanisms that Wnt4-modified NSC transplantation is sufficient to repair the injured spinal cord and recover the motor dysfunction after SCI.

Inhibition of Notch1 signaling promotes neuronal differentiation and improves functional recovery in spinal cord injury through suppressing the activation of Ras homolog family member A.

Neural stem cells (NSCs) transplantation represents a promising strategy for the repair of injured neurons, since NSCs not only produce multiple neurotrophic growth factors but also differentiate into mature cells to replace damaged cells. Previous studies have shown that Notch signaling pathway had negative effects on neuronal differentiation; however, the precise mechanism remained inadequately understood. This research aimed to investigate whether inhibition of Notch1 signaling promotes neuronal differentiation and improves functional recovery in rat spinal cord injury through suppressing the activation of Ras homolog family member A (RhoA). QPCR, western blot, and immunofluorescence experiments were used to analyze Notch1 signaling pathways, RhoA, Ras homologous -associated coiled-coil containing protein kinase 1 (ROCK1), cleaved caspased-3, and neuronal/astrocytic differentiation markers. The expression of RhoA and ROCK1 was inhibited by lentivirus or specific biochemical inhibitors. In spinal cord injury (SCI), motor function was assessed by hind limbs movements and electrophysiology. Tissue repairing was measured by immunofluorescence, Nissl staining, Fluorogold, HE staining, QPCR, western blot, and magnetic resonance imaging (MRI) experiments. Our results demonstrate that inhibition of Notch1 in NSCs can promote the differentiation of NSCs to neurons. Knockdown of RhoA and inhibition of ROCK1 both can promote neuronal differentiation through inhibiting the activation of Notch1 signaling pathway in NSCs. In SCI, silencing RhoA enhanced neuronal differentiation and improved tissue repairing/functional recovery by inhibiting the activation of Notch1 signaling pathway. Since Notch1 inhibits neuronal differentiation through activating the RhoA/ROCK1 signaling pathway in NSCs, our data suggest that the Notch1/RhoA/ROCK1/Hes1/Hes5 signaling pathway may serve as a novel target for the treatment of SCI.

Targeted inhibition of STAT3 in neural stem cells promotes neuronal differentiation and functional recovery in rats with spinal cord injury.

STAT3 is expressed in neural stem cells (NSCs), where a number of studies have previously shown that STAT3 is involved in regulating NSC differentiation. However, the possible molecular mechanism and role of STAT3 in spinal cord injury (SCI) remain unclear. In the present study, the potential effect of STAT3 in NSCs was first investigated by using short hairpin RNA (shRNA)-mediated STAT3 knockdown in rat NSCs in vitro. Immunofluorescence of ß3-tubulin and glial fibrillary acidic protein staining and western blotting showed that knocking down STAT3 expression promoted NSC neuronal differentiation, where the activity of mTOR was upregulated. Subsequently, rats underwent laminectomy and complete spinal cord transection followed by transplantation of NSCs transfected with control-shRNA or STAT3-shRNA at the injured site in vivo. Spinal cord-evoked potentials and the Basso-Beattie-Bresnahan scores were used to examine functional recovery. In addition, axonal regeneration and tissue repair were assessed using retrograde tracing with FluoroGold, hematoxylin and eosin, Nissl and immunofluorescence staining of ß3-tubulin, glial fibrillary acidic protein and microtubule-associated protein 2 following SCI. The results showed that transplantation with NSCs transfected with STAT3-RNA enhanced functional recovery following SCI and promoted tissue repair in rats, in addition to improving neuronal differentiation of the transplanted NSCs in the injury site. Taken together, in vitro and in vivo evidence that inhibiting STAT3 could promote NSC neuronal differentiation was demonstrated in the present study. Therefore, transplantation with NSCs with STAT3 expression knocked down appears to hold promising potential for enhancing the benefit of NSC-mediated regenerative cell therapy for SCI.

Transplantation of Wnt5a-modified NSCs promotes tissue repair and locomotor functional recovery after spinal cord injury.

Traditional therapeutic strategies for spinal cord injury (SCI) are insufficient to repair locomotor function because of the failure of axonal reconnection and neuronal regeneration in the injured central nervous system (CNS). Neural stem cell (NSC) transplantation has been considered a potential strategy and is generally feasible for repairing the neural circuit after SCI; however, the most formidable problem is that the neuronal differentiation rate of NSCs is quite limited. Therefore, it is essential to induce the neuronal differentiation of NSCs and improve the differentiation rate of NSCs in spinal cord repair. Our results demonstrate that both Wnt5a and miRNA200b-3p could promote NSC differentiation into neurons and that Wnt5a upregulated miRNA200b-3p expression through MAPK/JNK signaling to promote NSC differentiation into neurons. Wnt5a could reduce RhoA expression by upregulating miRNA200b-3p expression to inhibit activation of the RhoA/Rock signaling pathway, which has been reported to suppress neuronal differentiation. Overexpression of RhoA abolished the neurogenic capacity of Wnt5a and miRNA200b-3p. In vivo, miRNA200b-3p was critical for Wnt5a-induced NSC differentiation into neurons to promote motor functional and histological recovery after SCI by suppressing RhoA/Rock signaling. These findings provide more insight into SCI and help with the identification of novel treatment strategies.

Lentiviral vector delivery of short hairpin RNA to NgR1 promotes nerve regeneration and locomotor recovery in injured rat spinal cord.

Nogo receptor 1 (NgR1) is a high-affinity receptor of myelin-associated inhibitors (MAIs), and suppresses neurogenesis. Lentiviral vector are commonly used to alter the expression of targeted genes. However, little is known about the potential function of lentiviral vector harboring NgR1 shRNA (LV-NgR1 shRNA) on neurogenesis in spinal cord injury (SCI). In this study, the rats were randomly divided into three groups: including the LN (LV-NgR1 shRNA injection), LC (LV-control shRNA injection) and Sham (laminectomy only). Eight weeks post-injection of LV, spinal cords were examined by histology for changes in cavity size and by immunohistochemistry for changes in expression of NgR1, cell apoptosis, astrocytes, neurons and myelination. Motor function was assessed using the Basso, Beattie and Bresnahan (BBB) locomotor scale. Animals that received LV-NgR1 shRNA remarkably improved the motor function. These animals also showed an increase in levels of nerve fibers, synapses and myelination, a decrease in levels of lesion cavity and cell apoptosis at 8 weeks post-treatment. These findings give evidence that NgR1 may be a promising target for SCI treatment.

Tolerance limit of rats to normothermic hepatic inflow occlusion under portal blood bypass.

OBJECTIVE: To evaluate the tolerance limit of rats to normothermic hepatic inflow occlusion under portal blood bypass. METHODS: A new rat model of normothermic hepatic inflow occlusion under portal blood bypass was established by clamping temporarily the pedicles of all liver lobes while the caudal lobe was kept as a passage of the portal blood flow. After hepatic blood flow restored, the caudal lobe was cut off. On the 7th postoperative day, survival rate, hepatic morphological changes, and the severity and reversibility of the injured energy metabolism of the liver were investigated. RESULTS: All rats that had been subjected to 30, 60 and 90 minutes of hepatic inflow occlusion under portal blood bypass survived on the 7th postoperative day. ischemia-reperfusion injury of the liver was reversible and compensatory in rats with hepatic inflow occlusion within 90 minutes. However, the survival rates of rats with 100, 110 and 120 minutes of hepatic inflow occlusion were 50%, 30% and 20% respectively. Liver injury of rats with 120 minutes of hepatic inflow occlusion was severe and Irreversible. CONCLUSIONS: The tolerance limit of rats to normothermic hepatic inflow occlusion is enhanced significantly under portal blood bypass and the upper limit is 90 minutes.

Effect of aging on cytoskeleton system of Kupffer cell and its phagocytic capacity.

AIM:To investigate the age-related alterations of cytoskeleton system in liver Kupffer cell and their relation to the changed phagocytic function.METHODS: The phagocytic function of Kupffer cells from rats of various ages (6mo, 12mo,18mo and 24mo) were quantitatively evaluated by phagocytosis of polystyrene beads. The actin distribution and measurement of Kupffer cell were determined by a phalloidin-TRITC method; and the myosin and vimentin distribution and measurement with indirect immunochemical staining.RESULTS: Aging resulted in significant alterations of actin, myosin and vimentin distributions and reductions in Kupffer cell; the 3 cytoskeleton components of 24-mo-old Kupffer cell were significantly decreased to 68.0%, 84.9% and 75.5%, respectively of these of 6-mo-old Kupffer cell(P < 0.01,0.01 and 0.01). And these decreases had significant positive relations with the damaged phagocytosis of the aged Kupffer cell.gammavalues were 0.96(P < 0.05), 0.99(P < 0.01) and 0.95 (P < 0.05) respectively.CONCLUSION: The cytos-keleton system of the aged Kupffer cell presents an evident state of senescence, which may be an important mechanism of decreased phagocytosis of the aged Kupffer cell.