Changes in the Proteome of the Circle of Willis during Aging Reveal Signatures of Vascular Disease.

Approximately 70% of all strokes occur in patients over 65 years old, and stroke increases the risk of developing dementia. The circle of Willis (CoW), the ring of arteries at the base of the brain, links the intracerebral arteries to one another to maintain adequate cerebral perfusion. The CoW proteome is affected in cerebrovascular and neurodegenerative diseases, but changes related to aging have not been described. Here, we report on a quantitative proteomics analysis comparing the CoW from five young (2-3-month-old) and five aged male (18-20-month-old) mice using gene ontology (GO) enrichment, ingenuity pathway analysis (IPA), and iPathwayGuide tools. This revealed 242 proteins that were significantly dysregulated with aging, among which 189 were upregulated and 53 downregulated. GO enrichment-based analysis identified blood coagulation as the top biological function that changed with age and integrin binding and extracellular matrix constituents as the top molecular functions. Consistent with these findings, iPathwayGuide-based impact analysis revealed associations between aging and the complement and coagulation, platelet activation, ECM-receptor interaction, and metabolic process pathways. Furthermore, IPA analysis revealed the enrichment of 97 canonical pathways that contribute to inflammatory responses, as well as 59 inflammation-associated upstream regulators including 39 transcription factors and 20 cytokines. Thus, aging-associated changes in the CoW proteome in male mice demonstrate increases in metabolic, thrombotic, and inflammatory processes.

Short-Term Statin Treatment Reduces, and Long-Term Statin Treatment Abolishes, Chronic Vascular Injury by Radiation Therapy.

BACKGROUND: The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. We examined whether irradiation causes chronic vascular injury and whether short-term administration of statins during and after irradiation is sufficient to prevent chronic injury compared with long-term administration. METHODS AND RESULTS: C57Bl/6 mice were pretreated with pravastatin for 72 hours and then exposed to 12 Gy X-ray head-and-neck irradiation. Pravastatin was then administered either for an additional 24 hours or for 1 year. Carotid arteries were tested for vascular reactivity, altered gene expression, and collagen deposition 1 year after irradiation. Treatment with pravastatin for 24 hours after irradiation reduced the loss of endothelium-dependent vasorelaxation and protected against enhanced vasoconstriction. Expression of markers associated with inflammation (NFκB p65 [phospho-nuclear factor kappa B p65] and TNF-α [tumor necrosis factor alpha]) and with oxidative stress (NADPH oxidases 2 and 4) were lowered and subunits of the voltage and Ca2+ activated K+ BK channel (potassium calcium-activated channel subfamily M alpha 1 and potassium calcium-activated channel subfamily M regulatory beta subunit 1) in the carotid artery were modulated. Treatment with pravastatin for 1 year after irradiation completely reversed irradiation-induced changes. CONCLUSIONS: Short-term administration of pravastatin is sufficient to reduce chronic vascular injury at 1 year after irradiation. Long-term administration eliminates the effects of irradiation. These findings suggest that a prospective treatment strategy involving statins could be effective in patients undergoing radiation therapy. The optimal duration of treatment in humans has yet to be determined.

ATF4-dependent increase in mitochondrial-endoplasmic reticulum tethering following OPA1 deletion in skeletal muscle.

Mitochondria and endoplasmic reticulum (ER) contact sites (MERCs) are protein- and lipid-enriched hubs that mediate interorganellar communication by contributing to the dynamic transfer of Ca2+, lipid, and other metabolites between these organelles. Defective MERCs are associated with cellular oxidative stress, neurodegenerative disease, and cardiac and skeletal muscle pathology via mechanisms that are poorly understood. We previously demonstrated that skeletal muscle-specific knockdown (KD) of the mitochondrial fusion mediator optic atrophy 1 (OPA1) induced ER stress and correlated with an induction of Mitofusin-2, a known MERC protein. In the present study, we tested the hypothesis that Opa1 downregulation in skeletal muscle cells alters MERC formation by evaluating multiple myocyte systems, including from mice and Drosophila, and in primary myotubes. Our results revealed that OPA1 deficiency induced tighter and more frequent MERCs in concert with a greater abundance of MERC proteins involved in calcium exchange. Additionally, loss of OPA1 increased the expression of activating transcription factor 4 (ATF4), an integrated stress response (ISR) pathway effector. Reducing Atf4 expression prevented the OPA1-loss-induced tightening of MERC structures. OPA1 reduction was associated with decreased mitochondrial and sarcoplasmic reticulum, a specialized form of ER, calcium, which was reversed following ATF4 repression. These data suggest that mitochondrial stress, induced by OPA1 deficiency, regulates skeletal muscle MERC formation in an ATF4-dependent manner.

Radiation effects on retinal layers revealed by OCT, OCT-A, and perimetry as a function of dose and time from treatment.

Optical coherence tomography (OCT) has become a key method for diagnosing and staging radiation retinopathy, based mainly on the presence of fluid in the central macula. A robust retinal layer segmentation method is required for identification of the specific layers involved in radiation-induced pathology in individual eyes over time, in order to determine damage driven by radiation injury to the microvessels and to the inner retinal neurons. Here, we utilized OCT, OCT-angiography, visual field testing, and patient-specific dosimetry models to analyze abnormal retinal layer thickening and thinning relative to microvessel density, visual function, radiation dose, and time from radiotherapy in a cross-sectional cohort of uveal melanoma patients treated with 125I-plaque brachytherapy. Within the first 24 months of radiotherapy, we show differential thickening and thinning of the two inner retinal layers, suggestive of microvessel leakage and neurodegeneration, mostly favoring thickening. Four out of 13 eyes showed decreased inner retinal capillary density associated with a corresponding normal inner retinal thickness, indicating early microvascular pathology. Two eyes showed the opposite: significant inner retinal layer thinning and normal capillary density, indicating early neuronal damage preceding a decrease in capillary density. At later time points, inner retinal thinning becomes the dominant pathology and correlates significantly with decreased vascularity, vision loss, and dose to the optic nerve. Stable multiple retinal layer segmentation provided by 3D graph-based methods aids in assessing the microvascular and neuronal response to radiation, information needed to target therapeutics for radiation retinopathy and vision loss.

Mouse model of radiation retinopathy reveals vascular and neuronal injury.

PURPOSE: To characterize the neuronal and vascular pathology in vivo and in vitro in a mouse model of radiation retinopathy. METHODS: C57Bl/6J mice underwent cranial irradiation with 12 Gy and in vivo imaging by optical coherence tomography and of relative blood flow velocity by laser speckle flowgraphy for up to 3-6 months after irradiation. Retinal architecture, vascular density and leakage and apoptosis were analyzed by histology and immunohistochemistry before irradiation or at 10, 30, 240, and 365 days after treatment. RESULTS: The vascular density decreased in the plexiform layers starting at 30 days after irradiation. No impairment in retinal flow velocity was seen. Subtle perivascular leakage was present at 10 days, in particular in the outer plexiform layer. This corresponded to increased width of this layer. However, no significant change in the retinal thickness was detected by OCT-B scans. At 365 days after irradiation, the nuclear density was significantly reduced compared to baseline. Apoptosis was detected at 30 days and less prominent at 365 days. CONCLUSIONS: By histology, vascular leakage at 10 days was followed by increased neuronal apoptosis and loss of neuronal and vascular density. However, in vivo imaging approaches that are commonly used in human patients did not detect pathology in mice.

Short-term statin treatment reduces, and long-term statin treatment abolishes chronic vascular injury by radiation therapy.

Background: The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. Objectives: Determine if irradiation causes chronic vascular injury and whether short-term administration of statins during and after irradiation is sufficient to prevent chronic injury compared to long-term administration. Methods: C57Bl/6 mice were pretreated with pravastatin for 72 hours and then exposed to 12 Gy x-ray head-and-neck irradiation. Subsequently, they received pravastatin either for one additional day or for one year. Carotid arteries were tested for vascular reactivity and altered gene expression one year after irradiation. Results: Treatment with pravastatin for 24 hours reduced the loss of endothelium-dependent vasorelaxation and protected against enhanced vasoconstriction after IR. It reduced the expression of some markers associated with inflammation and oxidative stress and modulated that of subunits of the voltage and Ca2+ activated K+ (BK) channel in the carotid artery one year after irradiation. Treatment with pravastatin for one year completely reversed the changes caused by irradiation. Conclusions: In mice, short-term administration of pravastatin is sufficient to reduce chronic vascular injury after irradiation. Long-term administration eliminates the effects of irradiation. These findings suggest that a prospective treatment strategy involving statins could be effective in patients undergoing radiation therapy. The optimal duration of treatment in humans has yet to be determined.

Regulation of Smooth Muscle Cell Proliferation by Mitochondrial Ca2+ in Type 2 Diabetes.

Type 2 diabetes (T2D) is associated with increased risk of atherosclerotic vascular disease due to excessive vascular smooth muscle cell (VSMC) proliferation. Here, we investigated the role of mitochondrial dysfunction and Ca2+ levels in VSMC proliferation in T2D. VSMCs were isolated from normoglycemic and T2D-like mice induced by diet. The effects of mitochondrial Ca2+ uptake were studied using mice with selectively inhibited mitochondrial Ca2+/calmodulin-dependent kinase II (mtCaMKII) in VSMCs. Mitochondrial transition pore (mPTP) was blocked using ER-000444793. VSMCs from T2D compared to normoglycemic mice exhibited increased proliferation and baseline cytosolic Ca2+ levels ([Ca2+]cyto). T2D cells displayed lower endoplasmic reticulum Ca2+ levels, reduced mitochondrial Ca2+ entry, and increased Ca2+ leakage through the mPTP. Mitochondrial and cytosolic Ca2+ transients were diminished in T2D cells upon platelet-derived growth factor (PDGF) administration. Inhibiting mitochondrial Ca2+ uptake or the mPTP reduced VSMC proliferation in T2D, but had contrasting effects on [Ca2+]cyto. In T2D VSMCs, enhanced activation of Erk1/2 and its upstream regulators was observed, driven by elevated [Ca2+]cyto. Inhibiting mtCaMKII worsened the Ca2+ imbalance by blocking mitochondrial Ca2+ entry, leading to further increases in [Ca2+]cyto and Erk1/2 hyperactivation. Under these conditions, PDGF had no effect on VSMC proliferation. Inhibiting Ca2+-dependent signaling in the cytosol reduced excessive Erk1/2 activation and VSMC proliferation. Our findings suggest that altered Ca2+ handling drives enhanced VSMC proliferation in T2D, with mitochondrial dysfunction contributing to this process.

Mechanisms by which statins protect endothelial cells from radiation-induced injury in the carotid artery.

Background: The incidental use of statins during radiation therapy has been associated with a reduced long-term risk of developing atherosclerotic cardiovascular disease. However, the mechanisms by which statins protect the vasculature from irradiation injury remain poorly understood. Objectives: Identify the mechanisms by which the hydrophilic and lipophilic statins pravastatin and atorvastatin preserve endothelial function after irradiation. Methods: Cultured human coronary and umbilical vein endothelial cells irradiated with 4 Gy and mice subjected to 12 Gy head-and-neck irradiation were pretreated with statins and tested for endothelial dysfunction, nitric oxide production, oxidative stress, and various mitochondrial phenotypes at 24 and 240 h after irradiation. Results: Both pravastatin (hydrophilic) and atorvastatin (lipophilic) were sufficient to prevent the loss of endothelium-dependent relaxation of arteries after head-and-neck irradiation, preserve the production of nitric oxide by endothelial cells, and suppress the cytosolic reactive oxidative stress associated with irradiation. However, only pravastatin inhibited irradiation-induced production of mitochondrial superoxide; damage to the mitochondrial DNA; loss of electron transport chain activity; and expression of inflammatory markers. Conclusions: Our findings reveal some mechanistic underpinnings of the vasoprotective effects of statins after irradiation. Whereas both pravastatin and atorvastatin can shield from endothelial dysfunction after irradiation, pravastatin additionally suppresses mitochondrial injury and inflammatory responses involving mitochondria. Clinical follow-up studies will be necessary to determine whether hydrophilic statins are more effective than their lipophilic counterparts in reducing the risk of cardiovascular disease in patients undergoing radiation therapy.

The mitochondrial regulation of smooth muscle cell proliferation in type 2 diabetes.

Background: Type 2 diabetes (T2D) is associated with a strongly increased risk for restenosis after angioplasty driven by proliferation of vascular smooth muscle cells (VSMCs). Here, we sought to determine whether and how mitochondrial dysfunction in T2D drives VSMC proliferation with a focus on ROS and intracellular [Ca 2+ ] that both drive cell proliferation, occur in T2D and are regulated by mitochondrial activity. Methods: Using a diet-induced mouse model of T2D, the inhibition of the mitochondrial Ca 2+ /calmodulin-dependent kinase II (mtCaMKII), a regulator of Ca 2+ entry via the mitochondrial Ca 2+ uniporter selectively in VSMCs, we performed in vivo phenotyping after mechanical injury and established the mechanisms of excessive proliferation in cultured VSMCs. Results: In T2D, the inhibition of mtCaMKII reduced both neointima formation after mechanical injury and the proliferation of cultured VSMCs. VSMCs from T2D mice displayed accelerated proliferation, reduced mitochondrial Ca 2+ entry and membrane potential with elevated baseline [Ca 2+ ] cyto compared to cells from normoglycemic mice. Accelerated proliferation after PDGF treatment was driven by activation of Erk1/2 and its upstream regulators. Hyperactivation of Erk1/2 was Ca 2+ -dependent rather than mitochondrial ROS-driven Ca 2+ -dependent and included the activation of CaMKII in the cytosol. The inhibition of mtCaMKII exaggerated the Ca 2+ imbalance by lowering mitochondrial Ca 2+ entry and increasing baseline [Ca 2+ ] cyto , further enhancing baseline Erk1/2 activation. With inhibition of mtCaMKII, PDGF treatment had no additional effect on cell proliferation. Inhibition of activated CaMKII in the cytosol decreased excessive Erk1/2 activation and reduced VSMC proliferation. Conclusions: Collectively, our results provide evidence for the molecular mechanisms of enhanced VSMC proliferation after mechanical injury by mitochondrial Ca 2+ entry in T2D.

The BBSome regulates mitochondria dynamics and function.

OBJECTIVE: The essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function. METHODS: We used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1). RESULTS: Our data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS. CONCLUSIONS: These findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.

Cardiac Adverse Events Associated With Chemo-Radiation Versus Chemotherapy for Resectable Stage III Non-Small-Cell Lung Cancer: A Surveillance, Epidemiology and End Results-Medicare Study.

Background We compared cardiac outcomes for surgery-eligible patients with stage III non-small-cell lung cancer treated adjuvantly or neoadjuvantly with chemotherapy versus chemo-radiation therapy in the Surveillance, Epidemiology and End Results-Medicare database. Methods and Results Patients were age 66+, had stage IIIA/B resectable non-small-cell lung cancer diagnosed between 2007 and 2015, and received adjuvant or neoadjuvant chemotherapy or chemo-radiation within 121 days of diagnosis. Patients having chemo-radiation and chemotherapy only were propensity-score matched and followed from day 121 to first cardiac outcome, noncardiac death, radiation initiation by patients who received chemotherapy only, fee-for-service enrollment interruption, or December 31, 2016. Cause-specific hazard ratios (HRs) and competing risks subdistribution HRs were estimated. The primary outcome was the first of these severe cardiac events: acute myocardial infarction, other hospitalized ischemic heart disease, hospitalized heart failure, percutaneous coronary intervention/coronary artery bypass graft, cardiac death, or urgent/inpatient care for pericardial disease, conduction abnormality, valve disorder, or ischemic heart disease. With median follow-up of 13 months, 70 of 682 patients who received chemo-radiation (10.26%) and 43 of 682 matched patients who received chemotherapy only (6.30%) developed a severe cardiac event (P=0.008) with median time to first event 5.45 months. Chemo-radiation increased the rate of severe cardiac events (cause-specific HR: 1.62 [95% CI, 1.11-2.37] and subdistribution HR: 1.41 [95% CI, 0.97-2.04]). Cancer severity appeared greater among patients who received chemo-radiation (noncardiac death cause-specific HR, 2.53 [95% CI, 1.93-3.33] and subdistribution HR, 2.52 [95% CI, 1.90-3.33]). Conclusions Adding radiation therapy to chemotherapy is associated with an increased risk of severe cardiac events among patients with resectable stage III non-small-cell lung cancer for whom survival benefit of radiation therapy is unclear.

Mitochondrial Ca2+ Uptake Drives Endothelial Injury By Radiation Therapy.

BACKGROUND: Radiation therapy strongly increases the risk of atherosclerotic vascular disease, such as carotid stenosis. Radiation induces DNA damage, in particular in mitochondria, but the upstream and downstream signaling events are poorly understood. The objective of this study was to define such mechanisms. METHODS: Endothelial-specific MCU (mitochondrial Ca2+ uniporter) knockout and C57Bl6/J mice with or without a preinfusion of a mitoTEMPO (mitochondrial reactive oxygen species [ROS] scavenger) were exposed to a single dose of cranial irradiation. 24, and 240 hours postirradiation, vascular reactivity, endothelial function, and mitochondrial integrity were assessed ex vivo and in vitro. RESULTS: In cultured human endothelial cells, irradiation with 4 Gy increased cytosolic Ca2+ transients and the mitochondrial Ca2+ concentration ([Ca2+]mt) and activated MCU. These outcomes correlated with increases in mitochondrial ROS (mtROS), loss of NO production, and sustained damage to mitochondrial but not nuclear DNA. Moreover, irradiation impaired activity of the ETC (electron transport chain) and the transcription of ETC subunits encoded by mitochondrial DNA (mtDNA). Knockdown or pharmacological inhibition of MCU blocked irradiation-induced mtROS production, mtDNA damage, loss of NO production, and impairment of ETC activity. Similarly, the pretreatment with mitoTEMPO, a scavenger of mtROS, reduced irradiation-induced Ca2+ entry, and preserved both the integrity of the mtDNA and the production of NO, suggesting a feed-forward loop involving [Ca2+]m and mtROS. Enhancement of DNA repair in mitochondria, but not in the nucleus, was sufficient to block prolonged mtROS elevations and maintain NO production. Consistent with the findings from cultured cells, in C57BL/6J mice, head and neck irradiation decreased endothelium-dependent vasodilation, and mtDNA integrity in the carotid artery after irradiation. These effects were prevented by endothelial knockout of MCU or infusion with mitoTEMPO. CONCLUSIONS: Irradiation-induced damage to mtDNA is driven by MCU-dependent Ca2+ influx and the generation of mtROS. Such damage leads to reduced transcription of mitochondrial genes and activity of the ETC, promoting sustained mtROS production that induces endothelial dysfunction. Our findings suggest that targeting MCU and mtROS might be sufficient to mitigate irradiation-induced vascular disease.

Retraction notice to "Inhibition of CaMKII in mitochondria preserves endothelial barrier function after irradiation" [Free Radic. Biol. Med. 146C (2020) 287-298].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors and Editor-in-Chief. Some of the data presented in Figure 6C, F and G of the above-titled paper were reported incorrectly in the published article. After being contacted by the Journal, the authors discovered an unintentional error in how the original data were analyzed that could affect the accuracy of the subsequent analysis. The raw data were incorrectly grouped in the analysis software, thereby altering the comparisons. Therefore the authors wish to retract the paper and will recollect and reanalyze the data appropriately. The authors apologize for any inconvenience.

Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca2+ Handling.

Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

Retraction notice to Corrigendum to "Inhibition of CaMKII in mitochondria preserves endothelial barrier function after irradiation" [Free Radical Biol. Med. 146, January (2020) 287-298].

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors and Editor-in-Chief. Some of the data presented in Figure 6C, F and G of the paper to which this corrigendum relates were reported incorrectly in the published article. After being contacted by the Journal, the authors discovered an unintentional error in how the original data were analyzed that could affect the accuracy of the subsequent analysis. The raw data were incorrectly grouped in the analysis software, thereby altering the comparisons. Therefore the authors wish to retract the paper and corrigendum and will recollect and reanalyze the data appropriately. The authors apologize for any inconvenience.

Knockout of Sorbin And SH3 Domain Containing 2 (Sorbs2) in Cardiomyocytes Leads to Dilated Cardiomyopathy in Mice.

Background Sorbin and SH3 domain containing 2 (Sorbs2) protein is a cytoskeletal adaptor with an emerging role in cardiac biology and disease; yet, its potential relevance to adult-onset cardiomyopathies remains underexplored. Sorbs2 global knockout mice display lethal arrhythmogenic cardiomyopathy; however, the causative mechanisms remain unclear. Herein, we examine Sorbs2 dysregulation in heart failure, characterize novel Sorbs2 cardiomyocyte-specific knockout mice (Sorbs2-cKO), and explore associations between Sorbs2 genetic variations and human cardiovascular disease. Methods and Results Bioinformatic analyses show myocardial Sorbs2 mRNA is consistently upregulated in humans with adult-onset cardiomyopathies and in heart failure models. We generated Sorbs2-cKO mice and report that they develop progressive systolic dysfunction and enlarged cardiac chambers, and they die with congestive heart failure at about 1 year old. After 3 months, Sorbs2-cKO mice begin to show atrial enlargement and P-wave anomalies, without dysregulation of action potential-associated ion channel and gap junction protein expressions. After 6 months, Sorbs2-cKO mice exhibit impaired contractility in dobutamine-treated hearts and skinned myofibers, without dysregulation of contractile protein expressions. From our comprehensive survey of potential mechanisms, we found that within 4 months, Sorbs2-cKO hearts have defective microtubule polymerization and compensatory upregulation of structural cytoskeletal and adapter proteins, suggesting that this early intracellular structural remodeling is responsible for contractile dysfunction. Finally, we identified genetic variants that associate with decreased Sorbs2 expression and human cardiac phenotypes, including conduction abnormalities, atrial enlargement, and dilated cardiomyopathy, consistent with Sorbs2-cKO mice phenotypes. Conclusions Our studies show that Sorbs2 is essential for maintaining structural integrity in cardiomyocytes, likely through strengthening the interactions between microtubules and other cytoskeletal proteins at cross-link sites.

Reduced blood flow by laser speckle flowgraphy after 125I-plaque brachytherapy for uveal melanoma.

BACKGROUND: To determine whether reductions in retinal and choroidal blood flow measured by laser speckle flowgraphy are detected after 125I-plaque brachytherapy for uveal melanoma. METHODS: In a cross-sectional study, retinal and choroidal blood flow were measured using laser speckle flowgraphy in 25 patients after treatment with 125I-plaque brachytherapy for uveal melanoma. Flow was analyzed in the peripapillary region by mean blur rate as well as in the entire image area with a novel superpixel-based method. Relationships between measures were determined by Spearman correlation. RESULTS: Significant decreases in laser speckle blood flow were observed in both the retinal and choroidal vascular beds of irradiated, but not fellow, eyes. Overall, 24 of 25 patients had decreased blood flow compared to their fellow eye, including 5 of the 6 patients imaged within the first 6 months following brachytherapy. A significant negative correlation between blood flow and time from therapy was present. CONCLUSIONS: Decreases in retinal and choroidal blood flow by laser speckle flowgraphy were detected within the first 6 months following brachytherapy. Reduced retinal and choroidal blood flow may be an early indicator of microangiographic response to radiation therapy.

Longitudinal optical coherence tomography angiography (OCT-A) in a patient with radiation retinopathy following plaque brachytherapy for uveal melanoma.

Purpose: Patients with choroidal melanoma treated with brachytherapy lose vision over time due to radiation retinopathy and optic neuropathy. Newer imaging modalities such as optical coherence tomography angiography (OCT-A) may provide further insight into the ultrastructural vascular changes that occur over time. We studied the progressive OCT-A derived reduction in capillary density that occurred in the macula and juxtapapillary region of a patient treated with plaque brachytherapy for posterior uveal melanoma. Methods: A patient with medium-sized choroidal melanoma in the inferonasal mid-periphery of the right eye was followed with OCT-A imaging in addition to standard imaging (color fundus photography, standardized echography, OCT) over a four-year time period following brachytherapy. Images were analyzed to measure vascular density in nine discrete areas of the macula at each time point as a function of region-specific radiation dose. Results: OCT-A over time showed focal capillary loss and enlargement of the foveal avascular zone in addition to vascular re-modeling. These changes progressed over time despite improvement in the clinical markers of radiation retinopathy (cotton wool spots, retinal hemorrhages). Radiation dose significantly correlated with rate of reduction in vascular density assessed within 9 square sectors of the macula, and was greatest in sectors closest to the plaque, which had received the highest radiation dose. There was no change in the choriocapillaris flow area over time. The patient developed cystoid macular edema, but maintained 20/30 vision. Conclusions and Importance: Longitudinal OCT-A demonstrates the microvascular changes that occur in response to radiation over time. Identification of these features may help define therapeutic windows to prevent vision loss associated with radiation retinopathy and optic neuropathy. Ongoing studies will describe a larger cohort of patients followed with this modality over time.

Measuring hyperemic response to light flicker stimulus using continuous laser speckle flowgraphy in mice.

Alterations in neurovascular coupling have been associated with various ocular, cerebral, and systemic vascular disorders. In the eye, changes in vessel caliber by dynamic vessel analysis have been used to measure neurovascular coupling following a light flicker stimulus. Here, we present a new protocol for quantifying light-flicker induced hyperemia in the C57/Bl6J mouse retina using laser speckle flowgraphy (LSFG). Our protocol was adapted from protocols used in human subjects. By acquiring continuous time series data, we detected significant increase in blood flow. These responses are maintained with low variability over multiple imaging sessions, indicating these methods may be applied in serial studies of neurovascular coupling.