Spinal deformity surgery in patients for whom blood transfusion is not an option: a single-center experience.

OBJECTIVE: Spinal deformity surgery is associated with significant blood loss, often requiring the transfusion of blood and/or blood products. For patients declining blood or blood products, even in the face of life-threatening blood loss, spinal deformity surgery has been associated with high rates of morbidity and mortality. For these reasons, patients for whom blood transfusion is not an option have historically been denied spinal deformity surgery. METHODS: The authors retrospectively reviewed a prospectively collected data set. All patients declining blood transfusion who underwent spinal deformity surgery at a single institution between January 2002 and September 2021 were identified. Demographics collected included age, sex, diagnosis, details of any prior surgery, and medical comorbidities. Perioperative variables included levels decompressed and instrumented, estimated blood loss, blood conservation techniques used, length of surgery, length of hospital stay, and complications from surgery. Radiographic measurements included, where appropriate, sagittal vertical axis correction, Cobb angle correction, and regional angular correction. RESULTS: Spinal deformity surgery was performed in 31 patients (18 male, 13 female) over 37 admissions. The median age at surgery was 41.2 years (range 10.9-70.1 years), and 64.5% had significant medical comorbidities. A median of 9 levels (range 5-16 levels) were instrumented per surgery, and the median estimated blood loss was 800 mL (range 200-3000 mL). Posterior column osteotomies were performed in all surgeries, and pedicle subtraction osteotomies in 6 cases. Multiple blood conservation techniques were used in all patients. Preoperative erythropoietin was administered prior to 23 surgeries, intraoperative cell salvage was used in all, acute normovolemic hemodilution was performed in 20, and perioperative administration of antifibrinolytic agents was performed in 28 surgeries. No allogenic blood transfusions were administered. Surgery was staged intentionally in 5 cases, and there was 1 unintended staging due to intraoperative blood loss from a vascular injury. There was 1 readmission for a pulmonary embolus. There were 2 minor postoperative complications. The median length of stay was 6 days (range 3-28 days). Deformity correction and the goals of surgery were achieved in all patients. Two patients underwent revision surgery during the follow-up period: one for pseudarthrosis and the other for proximal junctional kyphosis. CONCLUSIONS: With proper preoperative planning and judicious use of blood conservation techniques, spinal deformity surgery may be performed safely in patients for whom blood transfusion is not an option. The same techniques can be applied widely to the general population in order to minimize blood loss and the need for allogeneic blood transfusion.

Immune checkpoint gene VSIR predicts patient prognosis in acute myeloid leukemia and myelodysplastic syndromes.

BACKGROUND: Immune checkpoint proteins play critical functions during the immune response to cancer and have been targeted by immune checkpoint blockade therapy. V-domain Ig suppressor of T cell activation (VSIR) is one of these immune checkpoint genes and has been investigated extensively in recent years due to its conflicting roles in cancer immunity. Specifically, in acute myeloid leukemia (AML), the prognostic value of VSIR is debated. RESULTS: In both patient tumor samples and cancer cell lines we find that VSIR has the highest expression in AML out of all cancer types and, in AML, has the highest expression out of all other immune checkpoint genes. Survival analysis indicated that AML patients with higher VSIR expression have significantly shorter survival than those patients with lower expression, even within established AML subgroups (e.g., FAB subtypes). Importantly, VSIR expression is predictive of progression from myelodysplastic syndromes (MDS) patients into AML, suggesting its potential role during the very early stage of AML development and progression. In addition to AML, VSIR also demonstrates prognostic values in other cancer types, including multiple myeloma and mesothelioma. CONCLUSION: In summary, our analyses revealed the prognostic value of VSIR and its potential as a target for immunotherapy, especially in AML.

Tumor cell intrinsic and extrinsic features predict prognosis in estrogen receptor positive breast cancer.

Although estrogen-receptor-positive (ER+) breast cancer is generally associated with favorable prognosis, clinical outcome varies substantially among patients. Genomic assays have been developed and applied to predict patient prognosis for personalized treatment. We hypothesize that the recurrence risk of ER+ breast cancer patients is determined by both genomic mutations intrinsic to tumor cells and extrinsic immunological features in the tumor microenvironment. Based on the Cancer Genome Atlas (TCGA) breast cancer data, we identified the 72 most common genomic aberrations (including gene mutations and indels) in ER+ breast cancer and defined sample-specific scores that systematically characterized the deregulated pathways intrinsic to tumor cells. To further consider tumor cell extrinsic features, we calculated immune infiltration scores for six major immune cell types. Many individual intrinsic features are predictive of patient prognosis in ER+ breast cancer, and some of them achieved comparable accuracy with the Oncotype DX assay. In addition, statistical learning models that integrated these features predicts the recurrence risk of patients with significantly better performance than the Oncotype DX assay (our optimized random forest model AUC = 0.841, Oncotype DX model AUC = 0.792, p = 0.04). As a proof-of-concept, our study indicates the great potential of genomic and immunological features in prognostic prediction for improving breast cancer precision medicine. The framework introduced in this work can be readily applied to other cancers.

A framework to predict the applicability of Oncotype DX, MammaPrint, and E2F4 gene signatures for improving breast cancer prognostic prediction.

To improve cancer precision medicine, prognostic and predictive biomarkers are critically needed to aid physicians in deciding treatment strategies in a personalized fashion. Due to the heterogeneous nature of cancer, most biomarkers are expected to be valid only in a subset of patients. Furthermore, there is no current approach to determine the applicability of biomarkers. In this study, we propose a framework to improve the clinical application of biomarkers. As part of this framework, we develop a clinical outcome prediction model (CPM) and a predictability prediction model (PPM) for each biomarker and use these models to calculate a prognostic score (P-score) and a confidence score (C-score) for each patient. Each biomarker's P-score indicates its association with patient clinical outcomes, while each C-score reflects the biomarker applicability of the biomarker's CPM to a patient and therefore the confidence of the clinical prediction. We assessed the effectiveness of this framework by applying it to three biomarkers, Oncotype DX, MammaPrint, and an E2F4 signature, which have been used for predicting patient response, pathologic complete response versus residual disease to neoadjuvant chemotherapy (a classification problem), and recurrence-free survival (a Cox regression problem) in breast cancer, respectively. In both applications, our analyses indicated patients with higher C scores were more likely to be correctly predicted by the biomarkers, indicating the effectiveness of our framework. This framework provides a useful approach to develop and apply biomarkers in the context of cancer precision medicine.

A B-Raf V600E gene signature for melanoma predicts prognosis and reveals sensitivity to targeted therapies.

BACKGROUND: B-Raf V600E mutations account for about half of all skin cutaneous melanoma cases, and patients with this mutation are sensitive to BRAF inhibitors. However, aberrations in other genes in the MAPK/ERK pathway may cascade a similar effect as B-Raf V600E mutations, rendering those patients sensitive to BRAF inhibitors. We rationalized that defining a signature based on B-Raf pathway activity may be more informative for prognosis and drug sensitivity prediction than a binary indicator such as mutation status. METHODS: In this study, we defined a B-Raf signature score using RNA-seq data from TCGA. A higher score is shown to not only predict B-Raf mutation status, but also predict other aberrations that could similarly activate the MAPK/ERK pathway, such as B-Raf amplification, RAS mutation, and EGFR amplification. RESULTS: We showed that patients dichotomized by the median B-Raf score is more significantly stratified than by other metrics of measuring B-Raf aberration, such as mutation status, gene expression, and protein expression. We also demonstrated that high B-Raf score predicts higher sensitivity to B-Raf inhibitors SB590885 and PLX4720, as expected, but also correlated with sensitivity to drugs targeting other relevant oncogenic pathways. CONCLUSION: The BRAF signature may better help guide targeted therapy for melanoma, and such a framework can be applied to other cancers and mutations to provide more information than mutation status alone.

A network-based integration for understanding racial disparity in prostate cancer.

Compared to Caucasians (CAs), African Americans (AAs) have a higher rate of incidence and mortality in prostate cancer and are prone to be diagnosed at later stages. To understand this racial disparity, molecular features of different types, including gene expression, DNA methylation and other genomic alterations, have been compared between tumor samples from the two races, but led to different disparity associated genes (DAGs). In this study, we applied a network-based algorithm to integrate a comprehensive set of genomic datasets and identified 130 core DAGs. Out of these genes, 78 were not identified by any individual dataset but prioritized and selected through network propagation. We found DAGs were highly enriched in several critical prostate cancer-related signaling transduction and cell cycle pathways and were more likely to be associated with patient prognosis in prostate cancer. Furthermore, DAGs were over-represented in prostate cancer risk genes identified from previous genome wide association studies. We also found DAGs were enriched in kinase and transcription factor encoding genes. Interestingly, for many of these prioritized kinases their association with racial disparity did not manifest from the original genomic/transcriptomic data but was reflected by their differential phosphorylation levels between AA and CA prostate tumor samples. Similarly, the disparity relevance of some transcription factors was not reflected at the mRNA or protein expression level, but at the activity level as demonstrated by their differential ability in regulating target gene expression. Our integrative analysis provided new candidate targets for improving prostate cancer treatment and addressing the racial disparity problem.

Computational modeling of chromatin accessibility identified important epigenomic regulators.

Chromatin accessibility is essential for transcriptional activation of genomic regions. It is well established that transcription factors (TFs) and histone modifications (HMs) play critical roles in chromatin accessibility regulation. However, there is a lack of studies that quantify these relationships. Here we constructed a two-layer model to predict chromatin accessibility by integrating DNA sequence, TF binding, and HM signals. By applying the model to two human cell lines (GM12878 and HepG2), we found that DNA sequences had limited power for accessibility prediction, while both TF binding and HM signals predicted chromatin accessibility with high accuracy. According to the HM model, HM features determined chromatin accessibility in a cell line shared manner, with the prediction power attributing to five core HM types. Results from the TF model indicated that chromatin accessibility was determined by a subset of informative TFs including both cell line-specific and generic TFs. The combined model of both TF and HM signals did not further improve the prediction accuracy, indicating that they provide redundant information in terms of chromatin accessibility prediction. The TFs and HM models can also distinguish the chromatin accessibility of proximal versus distal transcription start sites with high accuracy.

An Intelligent Augmented Lifelike Avatar App for Virtual Physical Examination of Suspected Strokes.

An intelligent-augmented lifelike avatar mobile app (iLAMA) that integrates computer vision and sensor readings to automate and streamline the NIH Stroke Scale (NIHSS) physical examination is presented. The user interface design is optimized for elderly patients while the app showcases an animated lifelike 3D model of a friendly physician who walks the user through the exam. The standardized NIHSS examination included in iLAMA consists of five core tasks. The first two tasks involve rolling the eyes to the left and then to the right, and then smiling as wide as the user can. The app determines facial landmarks and analyzes the palsy of the face. The next task is to extend the arm and hold the phone at the shoulder level, and the smart phone gyroscope is used to detect acceleration to determine possible weakness in the arm. Next, the app tracks the location of the hand keypoints and determines possible ataxia based on the precision and accuracy of the locations of the touches. Finally, the app determines the user's forward acceleration in walking and possible imbalances using the accelerometer. The app then sends analyzed results of these tasks to the neurologist or stroke specialist for review and decisions.Clinical Relevance- The physical examination of a stroke patient is a time consuming and repetitive process, and there is a lack of infrastructure and resource to monitor patient in post-stroke recovery after they leave the hospital for home or rehabilitation facilities. iLAMA app aims to automate a subset of the NIHSS physical examinations in measuring motor function recovery and also allows individual patients to track their performance over time. It will be an essential component in monitoring rehabilitation recovery and therapy effectiveness after hospitalization and can easily scaled to lo help millions of patients at a fraction of the cost.

Deep Learning Predicts EBV Status in Gastric Cancer Based on Spatial Patterns of Lymphocyte Infiltration.

EBV infection occurs in around 10% of gastric cancer cases and represents a distinct subtype, characterized by a unique mutation profile, hypermethylation, and overexpression of PD-L1. Moreover, EBV positive gastric cancer tends to have higher immune infiltration and a better prognosis. EBV infection status in gastric cancer is most commonly determined using PCR and in situ hybridization, but such a method requires good nucleic acid preservation. Detection of EBV status with histopathology images may complement PCR and in situ hybridization as a first step of EBV infection assessment. Here, we developed a deep learning-based algorithm to directly predict EBV infection in gastric cancer from H&E stained histopathology slides. Our model can not only predict EBV infection in gastric cancers from tumor regions but also from normal regions with potential changes induced by adjacent EBV+ regions within each H&E slide. Furthermore, in cohorts with zero EBV abundances, a significant difference of immune infiltration between high and low EBV score samples was observed, consistent with the immune infiltration difference observed between EBV positive and negative samples. Therefore, we hypothesized that our model's prediction of EBV infection is partially driven by the spatial information of immune cell composition, which was supported by mostly positive local correlations between the EBV score and immune infiltration in both tumor and normal regions across all H&E slides. Finally, EBV scores calculated from our model were found to be significantly associated with prognosis. This framework can be readily applied to develop interpretable models for prediction of virus infection across cancers.

Chr20q Amplification Defines a Distinct Molecular Subtype of Microsatellite Stable Colorectal Cancer.

Colorectal cancer is the third leading cause of cancer-related death in the United States. About 15% of colorectal cancers are associated with microsatellite instability (MSI) due to loss of function in the DNA mismatch repair pathway. This subgroup of patients has better survival rates and is more sensitive to immunotherapy. However, it remains unclear whether microsatellite stable (MSS) patients with colorectal cancer can be further stratified into subgroups with differential clinical characteristics. In this study, we analyzed The Cancer Genome Atlas data and found that Chr20q amplification is the most frequent copy number alteration that occurs specifically in colon (46%) and rectum (61%) cancer and is mutually exclusive with MSI. Importantly, MSS patients with Chr20q amplification (MSS-A) were associated with better recurrence-free survival compared with MSS patients without Chr20q amplification (MSS-N; P = 0.03). MSS-A tumors were associated with high level of chromosome instability and low immune infiltrations. In addition, MSS-A and MSS-N tumors were associated with somatic mutations in different driver genes, with high frequencies of mutated TP53 in MSS-A and mutated KRAS and BRAF in MSS-N. Our results suggest that MSS-A and MSS-N represent two subtypes of MSS colorectal cancer, and such stratification may be used to improve therapeutic treatment in an individualized manner. SIGNIFICANCE: This study shows that chromosome 20q amplification occurs predominately in microsatellite-stable colorectal cancer and defines a distinct subtype with good prognosis, high chromosomal instability, distinct mutation profiles, and low immune infiltrations.

Utility of intraoperative electromyography in placing C7 pedicle screws.

OBJECTIVE: The C7 vertebral body is morphometrically unique; it represents the transition from the subaxial cervical spine to the upper thoracic spine. It has larger pedicles but relatively small lateral masses compared to other levels of the subaxial cervical spine. Although the biomechanical properties of C7 pedicle screws are superior to those of lateral mass screws, they are rarely placed due to increased risk of neurological injury. Although pedicle screw stimulation has been shown to be safe and effective in determining satisfactory screw placement in the thoracolumbar spine, there are few studies determining its utility in the cervical spine. Thus, the purpose of this study was to determine the feasibility, clinical reliability, and threshold characteristics of intraoperative evoked electromyographic (EMG) stimulation in determining satisfactory pedicle screw placement at C7. METHODS: The authors retrospectively reviewed a prospectively collected data set. All adult patients who underwent posterior cervical decompression and fusion with placement of C7 pedicle screws at the authors' institution between January 2015 and March 2019 were identified. Demographic, clinical, neurophysiological, operative, and radiographic data were gathered. All patients underwent postoperative CT scanning, and the position of C7 pedicle screws was compared to intraoperative neurophysiological data. RESULTS: Fifty-one consecutive C7 pedicle screws were stimulated and recorded intraoperatively in 25 consecutive patients. Based on EMG findings, 1 patient underwent intraoperative repositioning of a C7 pedicle screw, and 1 underwent removal of a C7 pedicle screw. CT scans demonstrated ideal placement of the C7 pedicle screw in 40 of 43 instances in which EMG stimulation thresholds were > 15 mA. In the remaining 3 cases the trajectories were suboptimal but safe. When the screw stimulation thresholds were between 11 and 15 mA, 5 of 6 screws were suboptimal but safe, and in 1 instance was potentially dangerous. In instances in which the screw stimulated at thresholds ≤ 10 mA, all trajectories were potentially dangerous with neural compression. CONCLUSIONS: Ideal C7 pedicle screw position strongly correlated with EMG stimulation thresholds > 15 mA. In instances, in which the screw stimulates at values between 11 and 15 mA, screw trajectory exploration is recommended. Screws with thresholds ≤ 10 mA should always be explored, and possibly repositioned or removed. In conjunction with other techniques, EMG threshold testing is a useful and safe modality in determining appropriate C7 pedicle screw placement.

The case for T2 pedicle subtraction osteotomy in the surgical treatment of rigid cervicothoracic deformity.

OBJECTIVE: Rigid cervicothoracic kyphotic deformity (CTKD) remains a difficult pathology to treat, especially in the setting of prior cervical instrumentation and fusion. CTKD may result in chronic neck pain, difficulty maintaining horizontal gaze, and myelopathy. Prior studies have advocated for the use of C7 or T1 pedicle subtraction osteotomies (PSOs). However, these surgeries are fraught with danger and, most significantly, place the C7, C8, and/or T1 nerve roots at risk. METHODS: The authors retrospectively reviewed their experience with performing T2 PSO for the correction of rigid CTKD. Demographics collected included age, sex, details of prior cervical surgery, and coexisting conditions. Perioperative variables included levels decompressed, levels instrumented, estimated blood loss, length of surgery, length of stay, complications from surgery, and length of follow-up. Radiographic measurements included C2-7 sagittal vertical axis (SVA) correction, and changes in the cervicothoracic Cobb angle, lumbar lordosis, and C2-S1 SVA. RESULTS: Four male patients were identified (age range 55-72 years). Three patients had undergone prior posterior cervical laminectomy and instrumented fusion and developed postsurgical kyphosis. All patients underwent T2 PSO: 2 patients received instrumentation at C2-T4, and 2 patients received instrumentation at C2-T5. The median C2-7 SVA correction was 3.85 cm (range 2.9-5.3 cm). The sagittal Cobb angle correction ranged from 27.8° to 37.6°. Notably, there were no neurological complications. CONCLUSIONS: T2 PSO is a powerful correction technique for the treatment of rigid CTKD. Compared with C7 or T1 PSO, there is decreased risk of injury to intrinsic hand muscle innervators, and there is virtually no risk of vertebral artery injury. Laminectomy may also be safer, as there is less (or no) scar tissue from prior surgeries. Correction at this distal level may allow for a greater sagittal correction. The authors are optimistic that these findings will be corroborated in larger cohorts examining this challenging clinical entity.

Whole-genome deep-learning analysis identifies contribution of noncoding mutations to autism risk.

We address the challenge of detecting the contribution of noncoding mutations to disease with a deep-learning-based framework that predicts the specific regulatory effects and the deleterious impact of genetic variants. Applying this framework to 1,790 autism spectrum disorder (ASD) simplex families reveals a role in disease for noncoding mutations-ASD probands harbor both transcriptional- and post-transcriptional-regulation-disrupting de novo mutations of significantly higher functional impact than those in unaffected siblings. Further analysis suggests involvement of noncoding mutations in synaptic transmission and neuronal development and, taken together with previous studies, reveals a convergent genetic landscape of coding and noncoding mutations in ASD. We demonstrate that sequences carrying prioritized mutations identified in probands possess allele-specific regulatory activity, and we highlight a link between noncoding mutations and heterogeneity in the IQ of ASD probands. Our predictive genomics framework illuminates the role of noncoding mutations in ASD and prioritizes mutations with high impact for further study, and is broadly applicable to complex human diseases.

Minimally Invasive Evacuation of Spontaneous Cerebellar Intracerebral Hemorrhage.

OBJECTIVE: Spontaneous cerebellar intracerebral hemorrhage (scICH) constitutes ∼10% of all cases of spontaneous ICH, with a mortality of 20%-50%. Suboccipital craniectomy (SOC) is commonly performed for scICH causing brainstem compression or hydrocephalus. However, SOC requires long anesthesia times and results in a high complication rate. We present a series of patients who minimally invasive scICH evacuation as an alternative to traditional SOC. METHODS: We retrospectively reviewed the operative records for patients presenting to a single center from January 1, 2009 to March 1, 2017. All patients who had undergone evacuation of scICH were included in the present study. Clinical and radiographic variables were collected, including admission and postoperative Glasgow coma scale (GCS) scores, preoperative and postoperative hematoma volumes, and modified Rankin scale (mRS) scores at long-term follow-up. RESULTS: We identified 10 patients who had presented with scICH requiring surgery. All scICH evacuations were performed through a minicraniectomy positioned in the suboccipital area as close to the hematoma as possible. The mean patient age was 64.1 years. The mean presenting GCS score was 8.6, the mean initial hematoma volume was 25.4 mL, the mean procedure time was 57 minutes, and the mean postoperative hematoma volume was 2.8 mL. The mortality rate was 10% and mean long-term follow-up mRS score was 2. CONCLUSIONS: Minimally invasive scICH hematoma evacuation is a feasible alternative to SOC with numerous advantages that could lead to improved radiographic and clinical results.

Complications of Poor Cervical Alignment in Patients Undergoing Posterior Cervicothoracic Laminectomy and Fusion.

OBJECTIVE: This study sought to determine whether a relationship exists between caudal instrumented level and revision rates, neck disability index scores, and cervical alignment in patients undergoing multilevel posterior cervical fusion. METHODS: This study examined a dataset of all patients undergoing posterior cervical decompression and fusion at ≥3 levels, terminating between C4 and T4, between January 2010 and December 2015, with at least 12 months of clinical follow-up. Patients were separated into cohorts based on caudal level of the fusion: C6 (or more cranial), C7, T1, or T2 (or more caudal). Revision rate, neck disability index score, sagittal vertical axis, T1 slope, and cervical lordosis were recorded. Linear regression and multivariate analysis were performed to identify independent predictors of patient outcomes and disparities between ending constructs in the cervical and the thoracic spine. RESULTS: The overall revision rate was 10.8% (n = 24). No statistically significant difference in the revision rate was identified between fusions terminating at C6 or cranial, C7, T1, or T2 and caudal (P = 0.74). Revision correlated strongly with increased sagittal vertical axis (P = 0.002) and T1 slope (P = 0.04). Increased neck disability index score correlated with revision rate (P = 0.01), cervical kyphosis (P < 0.001), and increased sagittal vertical axis (P = 0.04). CONCLUSIONS: This study suggests that constructs terminating in the proximal thoracic spine have similar revision rates, postoperative neck disability index scores, and radiographic measurements as those terminating in the cervical spine. Poor cervical alignment, as evidenced by increased sagittal vertical axis, cervical kyphosis and T1 slope, predicts need for revision and of poorer clinical outcomes.

Deep learning sequence-based ab initio prediction of variant effects on expression and disease risk.

Key challenges for human genetics, precision medicine and evolutionary biology include deciphering the regulatory code of gene expression and understanding the transcriptional effects of genome variation. However, this is extremely difficult because of the enormous scale of the noncoding mutation space. We developed a deep learning-based framework, ExPecto, that can accurately predict, ab initio from a DNA sequence, the tissue-specific transcriptional effects of mutations, including those that are rare or that have not been observed. We prioritized causal variants within disease- or trait-associated loci from all publicly available genome-wide association studies and experimentally validated predictions for four immune-related diseases. By exploiting the scalability of ExPecto, we characterized the regulatory mutation space for human RNA polymerase II-transcribed genes by in silico saturation mutagenesis and profiled > 140 million promoter-proximal mutations. This enables probing of evolutionary constraints on gene expression and ab initio prediction of mutation disease effects, making ExPecto an end-to-end computational framework for the in silico prediction of expression and disease risk.

miR-378 reduces mesangial hypertrophy and kidney tubular fibrosis via MAPK signalling.

The regulatory role of a novel miRNA, miR-378, was determined in the development of fibrosis through repression of the MAPK1 pathway, miR-378 and fibrotic gene expression was examined in streptozotocin (STZ)-induced diabetic mice at 18 weeks or in unilateral ureteral obstruction (UUO) mice at 7 days. miR-378 transfection of proximal tubular epithelial cells, NRK52E and mesangial cells was assessed with/without endogenous miR-378 knockdown using the locked nucleic acid (LNA) inhibitor. NRK52E cells were co-transfected with the mothers against decapentaplegic homolog 3 (SMAD3) CAGA reporter and miR-378 in the presence of transforming growth factor-ß (TGF-ß1) was assessed. Quantitative polymerase chain reaction (qPCR) showed a significant reduction in miR-378 (P<0.05) corresponding with up-regulated type I collagen, type IV collagen and α-smooth muscle actin (SMA) in kidneys of STZ or UUO mice, compared with controls. TGF-ß1 significantly increased mRNA expression of type I collagen (P<0.05), type IV collagen (P<0.05) and α-SMA (P<0.05) in NRK52E cells, which was significantly reduced (P<0.05) following miR-378 transfection and reversed following addition of the LNA inhibitor of endogenous miR-378 Overexpression of miR-378 inhibited mesangial cell expansion and proliferation in response to TGF-ß1, with LNA-miR-378 transfection reversing this protective effect, associated with cell morphological alterations. The protective function of MAPK1 on miR-378 was shown in kidney cells treated with the MAPK1 inhibitor, selumetinib, which inhibited mesangial cell hypertrophy in response to TGF-ß1. Taken together, these results suggest that miR-378 acts via regulation of the MAPK1 pathway. These studies demonstrate the protective function of MAPK1, regulated by miR-378, in the induction of kidney cell fibrosis and mesangial hypertrophy.

Mesenchymal Stem Cells Deliver Exogenous MicroRNA-let7c via Exosomes to Attenuate Renal Fibrosis.

The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-ß1, and TGF-ß type 1 receptor (TGF-ßR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-ß1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-ßR1 confirmed that miR-let7c attenuates TGF-ß1-driven TGF-ßR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.

Mesenchymal stem cells as novel micro-ribonucleic acid delivery vehicles in kidney disease.

MicroRNAs (miRNAs) are short single strands of RNA responsible for post-transcriptional regulation of gene expression and have been implicated in the pathogenesis of chronic kidney disease (CKD). Emerging evidence reports that miRNAs can reduce kidney fibrosis through regulation of targets associated with collagen and extracellular matrix accumulation. However, the development of miRNA therapies has been hampered by the lack of targeted and sustainable methods of systemic miRNA delivery. Mesenchymal stem cells (MSCs) provide a promising miRNA delivery platform to overcome toxicity, the potential for insertional mutations and the low efficiency of previous methods. MSCs are endogenously immunoprivileged and home to sites of inflammation. They also release trophic growth factors to modulate the immune system, alter the polarization of macrophages and provide renal protection and repair. The potential to engineer MSCs to express or overexpress miRNAs, released by exosomes, may enhance their natural functions. Clinical studies are already being conducted individually for the use of miRNAs in cancer and MSCs in diseases associated with CKD. Hence, the combination of miRNAs and MSCs may provide an unparalleled cell-based therapy for treating CKD.