Brett Johnson

The Endothelial-Specific MicroRNA miR-126 Governs Vascular Integrity and Angiogenesis

Endothelial cells play essential roles in maintenance of vascular integrity, angiogenesis, and wound repair. We show that an endothelial cell-restricted microRNA (miR-126) mediates developmental angiogenesis in vivo. Targeted deletion of miR-126 in mice causes leaky vessels, hemorrhaging, and partial embryonic lethality, due to a loss of vascular integrity and defects in endothelial cell proliferation, migration, and angiogenesis. The subset of mutant animals that survives displays defective cardiac neovascularization following myocardial infarction. The vascular abnormalities of miR-126 mutant mice resemble the consequences of diminished signaling by angiogenic growth factors, such as VEGF and FGF. Accordingly, miR-126 enhances the proangiogenic actions of VEGF and FGF and promotes blood vessel formation by repressing the expression of Spred-1, an intracellular inhibitor of angiogenic signaling. These findings have important therapeutic implications for a variety of disorders involving abnormal angiogenesis and vascular leakage.

A Family of microRNAs Encoded by Myosin Genes Governs Myosin Expression and Muscle Performance

Myosin is the primary regulator of muscle strength and contractility. Here we show that three myosin genes, Myh6, Myh7, and Myh7b, encode related intronic microRNAs (miRNAs), which, in turn, control muscle myosin content, myofiber identity, and muscle performance. Within the adult heart, the Myh6 gene, encoding a fast myosin, coexpresses miR-208a, which regulates the expression of two slow myosins and their intronic miRNAs, Myh7/miR-208b and Myh7b/miR-499, respectively. miR-208b and miR-499 play redundant roles in the specification of muscle fiber identity by activating slow and repressing fast myofiber gene programs. The actions of these miRNAs are mediated in part by a collection of transcriptional repressors of slow myofiber genes. These findings reveal that myosin genes not only encode the major contractile proteins of muscle, but act more broadly to influence muscle function by encoding a network of intronic miRNAs that control muscle gene expression and performance.

miR-15 Family Regulates Postnatal Mitotic Arrest of Cardiomyocytes

Rationale: Mammalian cardiomyocytes withdraw from the cell cycle during early postnatal development, which significantly limits the capacity of the adult mammalian heart to regenerate after injury. The regulatory mechanisms that govern cardiomyocyte cell cycle withdrawal and binucleation are poorly understood. Objective: Given the potential of microRNAs (miRNAs) to influence large gene networks and modify complex developmental and disease phenotypes, we searched for miRNAs that were regulated during the postnatal switch to terminal differentiation. Methods and Results: Microarray analysis revealed subsets of miRNAs that were upregulated or downregulated in cardiac ventricles from mice at 1 and 10 days of age (P1 and P10). Interestingly, miR-195 (a member of the miR-15 family) was the most highly upregulated miRNA during this period, with expression levels almost 6-fold higher in P10 ventricles relative to P1. Precocious overexpression of miR-195 in the embryonic heart was associated with ventricular hypoplasia and ventricular septal defects in &bgr;-myosin heavy chain–miR-195 transgenic mice. Using global gene profiling and argonaute-2 immunoprecipitation approaches, we showed that miR-195 regulates the expression of a number of cell cycle genes, including checkpoint kinase 1 (Chek1), which we identified as a highly conserved direct target of miR-195. Finally, we demonstrated that knockdown of the miR-15 family in neonatal mice with locked nucleic acid–modified anti-miRNAs was associated with an increased number of mitotic cardiomyocytes and derepression of Chek1. Conclusions: These findings suggest that upregulation of the miR-15 family during the neonatal period may be an important regulatory mechanism governing cardiomyocyte cell cycle withdrawal and binucleation.

A cardiac MicroRNA governs systemic energy homeostasis by regulation of MED13

Obesity, type 2 diabetes, and heart failure are associated with aberrant cardiac metabolism. We show that the heart regulates systemic energy homeostasis via MED13, a subunit of the Mediator complex, which controls transcription by thyroid hormone and other nuclear hormone receptors. MED13, in turn, is negatively regulated by a heart-specific microRNA, miR-208a. Cardiac-specific overexpression of MED13 or pharmacologic inhibition of miR-208a in mice confers resistance to high-fat diet-induced obesity and improves systemic insulin sensitivity and glucose tolerance. Conversely, genetic deletion of MED13 specifically in cardiomyocytes enhances obesity in response to high-fat diet and exacerbates metabolic syndrome. The metabolic actions of MED13 result from increased energy expenditure and regulation of numerous genes involved in energy balance in the heart. These findings reveal a role of the heart in systemic metabolic control and point to MED13 and miR-208a as potential therapeutic targets for metabolic disorders.

MicroRNA-214 protects the mouse heart from ischemic injury by controlling Ca2+ overload and cell death

Early reperfusion of ischemic cardiac tissue remains the most effective intervention for improving clinical outcome following myocardial infarction. However, abnormal increases in intracellular Ca²⁺ during myocardial reperfusion can cause cardiomyocyte death and consequent loss of cardiac function, referred to as ischemia/reperfusion (IR) injury. Therapeutic modulation of Ca²⁺ handling provides some cardioprotection against the paradoxical effects of restoring blood flow to the heart, highlighting the significance of Ca²⁺ overload to IR injury. Cardiac IR is also accompanied by dynamic changes in the expression of microRNAs (miRNAs); for example, miR-214 is upregulated during ischemic injury and heart failure, but its potential role in these processes is unknown. Here, we show that genetic deletion of miR-214 in mice causes loss of cardiac contractility, increased apoptosis, and excessive fibrosis in response to IR injury. The cardioprotective roles of miR-214 during IR injury were attributed to repression of the mRNA encoding sodium/calcium exchanger 1 (Ncx1), a key regulator of Ca²⁺ influx; and to repression of several downstream effectors of Ca²⁺ signaling that mediate cell death. These findings reveal a pivotal role for miR-214 as a regulator of cardiomyocyte Ca²⁺ homeostasis and survival during cardiac injury.

TDP-43 is directed to stress granules by sorbitol, a novel physiological osmotic and oxidative stressor

ABSTRACT TDP-43, or TAR DNA-binding protein 43, is a pathological marker of a spectrum of neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. TDP-43 is an RNA/DNA-binding protein implicated in transcriptional and posttranscriptional regulation. Recent work also suggests that TDP-43 associates with cytoplasmic stress granules, which are transient structures that form in response to stress. In this study, we establish sorbitol as a novel physiological stressor that directs TDP-43 to stress granules in Hek293T cells and primary cultured glia. We quantify the association of TDP-43 with stress granules over time and show that stress granule association and size are dependent on the glycine-rich region of TDP-43, which harbors the majority of pathogenic mutations. Moreover, we establish that cells harboring wild-type and mutant TDP-43 have distinct stress responses: mutant TDP-43 forms significantly larger stress granules, and is incorporated into stress granules earlier, than wild-type TDP-43; in striking contrast, wild-type TDP-43 forms more stress granules over time, but the granule size remains relatively unchanged. We propose that mutant TDP-43 alters stress granule dynamics, which may contribute to the progression of TDP-43 proteinopathies.

Current opinion in urology 2017: Focal therapy of small renal lesions

Purpose of review With the increasing incidence of small renal masses (SRMs), ablative technologies are becoming more commonly utilized. With any nascent treatment modality, outcomes literature needs to be constantly re-evaluated. The purpose of this review is to revisit the most updated literature regarding the safety and efficacy of ablative treatments of renal lesions. Recent findings Recent literature demonstrates that small renal tumor ablation is safe and effective. Although it does not have the same oncological efficacy of surgical extirpation, local recurrence-free survival has consistently shown to be around 90%. Cryoablation and radiofrequency ablation have longer-term data demonstrating durable responses. Microwave ablation and irreversible electroporation are promising modalities with longer-term data coming. Complication rates and procedural morbidity of ablation are consistently lower than for partial nephrectomy. Summary Image-guided focal ablation is a valuable tool in the management of SRMs. Although it does not have the same efficacy of surgical extirpation, with the ability to perform repeat procedures and salvage surgery if necessary, oncologic outcomes are comparable to those of upfront surgery. Ultimately, longer-term studies and prospective trials are needed to further elucidate these modalities.

Surgical Outcomes of Three vs Four Arm Robotic Partial Nephrectomy: Is the Fourth Arm Necessary?

OBJECTIVE To compare the cost, efficacy, and safety of 3-arm versus 4-arm technique in robotic partial nephrectomy (RPN). Surgeons may either elect to utilize three vs four robotic instruments depending on preference. The purpose of this study is to compare the outcomes between the two techniques. METHODS RPNs from June 2016 to August 2017 were retrospectively reviewed. Tumor features, surgical parameters, and operative outcomes were evaluated. The number of arms used was determined. Statistical analysis was performed with the Students t test, chi-squared, and Mann-Whitney test. RESULTS A total of 61 consecutive 3-arm RPNs and 59 consecutive 4-arm RPNs were evaluated. Mean tumor diameter and median nephrometry score were 3.4 cm (± 1.1 SD) and 7 (6-8 IQR) for the 3-arm group and 3.3 cm (±1.2 SD) and 6 (5-8 IQR) for the 4-arm group, respectively (size: p = 0.7, nephrometry: p = 0.07). Hospital length of stay, operative time, estimated blood loss, complication rate, blood transfusion rate, and readmission rate all demonstrated no statistically significant difference between 3-arm and 4-arm groups (p >0.05). Mean ischemia time was shorter by 5.1 minutes in the 4-arm group (p = 0.02). Rate of margin positivity was higher in the 4-arm group (0% vs 10%, p = 0.03). CONCLUSION RPN can be safely and effectively completed with 3-robotic arms. While there was increased ischemia time, the difference was small and likely not of clinical significance. The routine addition of the fourth robotic arm in RPN is not necessary.

Ten-year outcomes of renal tumor radiofrequency ablation

PURPOSE We reviewed long-term oncologic outcomes in patients with renal tumors treated with radio frequency ablation more than 10 years ago. MATERIALS AND METHODS We retrospectively reviewed the records of patients with renal tumors who underwent radio frequency ablation from November 2000 to August 2007. Demographic, clinical and radiological data were assessed to determine evidence of disease recurrence. Patients with familial renal cell carcinoma syndromes were excluded from study. We calculated disease-free, metastasis-free, cancer specific and overall survival with the Kaplan-Meier method. Subgroup analysis of patients who had at least 10 years followup was performed to determine actual 10-year survival. Analysis was also performed based on tumor size. RESULTS A total of 112 tumors in 106 patients were treated with radio frequency ablation. Median followup was 79 months (IQR 28.9-121.1) and mean ± SD tumor size was 2.5 ± 0.8 cm. Initial technical success was achieved in 97% of cases. There were 10 recurrences. Kaplan-Meier 6-year disease-free and cancer specific survival rates were 89% and 96%, respectively. Disease-free survival decreased to 68% for tumors greater than 3 cm. In the subgroup with at least 10-year followup the actual disease-free, cancer specific and overall survival rates were 82%, 94% and 49%, respectively. No patient experienced recurrence after 5 years. CONCLUSIONS Radio frequency ablation is a safe and effective treatment option for small renal masses less than 3 cm in diameter. We report good oncologic outcomes with actual 10-year survival data. No recurrence developed after 5 years. Tumors greater than 3 cm have significantly poorer outcomes.Purpose: We reviewed long-term oncologic outcomes in patients with renal tumors treated with radio frequency ablation more than 10 years ago. Materials and Methods: We retrospectively reviewed the records of patients with renal tumors who underwent radio frequency ablation from November 2000 to August 2007. Demographic, clinical and radiological data were assessed to determine evidence of disease recurrence. Patients with familial renal cell carcinoma syndromes were excluded from study. We calculated disease-free, metastasis-free, cancer specific and overall survival with the Kaplan-Meier method. Subgroup analysis of patients who had at least 10 years followup was performed to determine actual 10-year survival. Analysis was also performed based on tumor size. Results: A total of 112 tumors in 106 patients were treated with radio frequency ablation. Median followup was 79 months (IQR 28.9–121.1) and mean ± SD tumor size was 2.5 ± 0.8 cm. Initial technical success was achieved in 97% of cases. There were 10 recurrences. Kaplan-Meier 6-year disease-free and cancer specific survival rates were 89% and 96%, respectively. Disease-free survival decreased to 68% for tumors greater than 3 cm. In the subgroup with at least 10-year followup the actual disease-free, cancer specific and overall survival rates were 82%, 94% and 49%, respectively. No patient experienced recurrence after 5 years. Conclusions: Radio frequency ablation is a safe and effective treatment option for small renal masses less than 3 cm in diameter. We report good oncologic outcomes with actual 10-year survival data. No recurrence developed after 5 years. Tumors greater than 3 cm have significantly poorer outcomes.

Development and evaluation of a novel endoscopic sack to facilitate tissue prostate adenoma morcellation

INTRODUCTION Morcellation of the adenoma after laser enucleation of the prostate (LEP) is both time-consuming and prone to complications. We have designed a novel polyethylene sack (ProSac) to improve the morcellation process following LEP. Both silicone and cadaver models were utilized to evaluate the safety and efficacy of ProSac. METHODS The inanimate model used tissue-mimicking silicone to accurately approximate bladder volume and compliance. The second model was developed using a fresh cadaver. Heat-fixed chicken breast was used to mimic enucleated prostatic adenoma. Morcellation of the simulated adenoma tissue was tested in both models with and without the ProSac. Morcellated tissue was removed from the filter, desiccated, and weighed after each run to establish morcellation efficiency. Visual inspection was used to evaluate for mucosal injury or bladder perforation. A hydromanometer was placed in the cadaveric bladder to measure bladder pressure. RESULTS The device was able to capture up to 30 g of tissue with good closure while maintaining good distention and visualization during morcellation. In the silicone model, morcellation efficiency with the device was 4.6 g/minute, while efficiency without the device was 2.6 g/minute (p = 0.03). In the cadaveric model, mean entrapment time was 22 ± 11 seconds. Morcellation efficiency with the device was 2.1 g/minute when excluding entrapment time and 1.9 g/minute including entrapment time. Without the ProSac, morcellation efficiency was 1.2 g/minute (p = 0.05). In both models, multiple mucosal injuries occurred without the device, while none occurred with the device. Bladder pressure was similar between study arms. CONCLUSIONS The ProSac is a novel device that can provide additional safety during adenoma morcellation. It may also achieve clinically and statistically significant improvement in morcellation efficiency without increasing bladder pressure.