A spatially specified systems pharmacology therapy for axonal recovery after injury.

There are no known drugs or drug combinations that promote substantial central nervous system axonal regeneration after injury. We used systems pharmacology approaches to model pathways underlying axonal growth and identify a four-drug combination that regulates multiple subcellular processes in the cell body and axons using the optic nerve crush model in rats. We intravitreally injected agonists HU-210 (cannabinoid receptor-1) and IL-6 (interleukin 6 receptor) to stimulate retinal ganglion cells for axonal growth. We applied, in gel foam at the site of nerve injury, Taxol to stabilize growing microtubules, and activated protein C to clear the debris field since computational models predicted that this drug combination regulating two subcellular processes at the growth cone produces synergistic growth. Physiologically, drug treatment restored or preserved pattern electroretinograms and some of the animals had detectable visual evoked potentials in the brain and behavioral optokinetic responses. Morphology experiments show that the four-drug combination protects axons or promotes axonal regrowth to the optic chiasm and beyond. We conclude that spatially targeted drug treatment is therapeutically relevant and can restore limited functional recovery.

Whole cell response to receptor stimulation involves many deep and distributed subcellular biochemical processes.

Neurite outgrowth is an integrated whole cell response triggered by the cannabinoid-1 receptor. We sought to identify the many different biochemical pathways that contribute to this whole cell response. To understand underlying mechanisms, we identified subcellular processes (SCPs) composed of one or more biochemical pathways and their interactions required for this response. Differentially expressed genes and proteins were obtained from bulk transcriptomics and proteomic analysis of extracts from cells stimulated with a cannabinoid-1 receptor agonist. We used these differentially expressed genes and proteins to build networks of interacting SCPs by combining the expression data with prior pathway knowledge. From these SCP networks, we identified additional genes that when ablated, experimentally validated the SCP involvement in neurite outgrowth. Our experiments and informatics modeling allowed us to identify diverse SCPs such as those involved in pyrimidine metabolism, lipid biosynthesis, and mRNA splicing and stability, along with more predictable SCPs such as membrane vesicle transport and microtubule dynamics. We find that SCPs required for neurite outgrowth are widely distributed among many biochemical pathways required for constitutive cellular functions, several of which are termed 'deep', since they are distal to signaling pathways and the key SCPs directly involved in extension of the neurite. In contrast, 'proximal' SCPs are involved in microtubule growth and membrane vesicle transport dynamics required for neurite outgrowth. From these bioinformatics and dynamical models based on experimental data, we conclude that receptor-mediated regulation of subcellular functions for neurite outgrowth is both distributed, that is, involves many different biochemical pathways, and deep.

Partial In-Stent Thrombosis After Iliac Vein Stenting in Non-Thrombotic Vein Lesions.

OBJECTIVE: Iliac vein stenting is a safe and efficacious procedure for the correction of iliac vein stenosis. One of its known major complications is complete iliac vein stent thrombosis. However, we have noticed in our practice that a number of patients developed only early partial in-stent (<60%) thrombosis, within the first 30 days. In order to try to learn more about these lesions, we reviewed the data for possible causes of these lesions. MATERIALS/METHODS: From September 2012 to August 2018, we obtained 3518 iliac vein venograms using intravascular ultrasound (IVUS) for patients with venous insufficiency who failed to respond to conservative therapy. Patients were followed up with transcutaneous duplex ultrasound (DUS) every 3 months for the first year and every 6 - 12 months thereafter. Patients were prescribed clopidogrel for 3 months or were told to continue their pre-existing anticoagulants. RESULTS: There were 2234 women and 1284 men who received an iliac vein stent. The mean age was 65.7 ±14 years. Mean follow-up for this cohort was 17 months. Of 74 patients developed a full thrombosis, 38 developed a partial venous thrombosis and 3406 developed no thrombosis. When comparing those who developed a partial thrombus versus those who developed no thrombus/full thrombus, overall age, laterality, CEAP, gender, and whether the patient received clopidogrel prior to the procedure and after the procedure were not found to be statistically significant factors. However, patients with an ASA score of 2 or 3,were found to be at a higher risk of developing a partial thrombus(P = 0.0223) compared to those who had an ASA score of 1 or 4. CEAP Scores and ASA class breakdown can be seen in Table 1 and Table 2, respectively. Of the 38 partial venous thrombosis that developed,18 completely resolved within the first 3 months after the procedure and 20 remained chronic past 3 months after the procedure. Patients with partial venous thrombosis were asymptomatic upon clinical presentation, and none developed post thrombotic syndrome (PTS) or pulmonary embolism (PE). Male gender was associated with partial thrombus resolution(P = 0.0036) CONCLUSIONS: Patients with ASA scores of 2 or 3, seemed to be at a higher risk of developing a partial thrombus when compared to patients with ASA score of 1 or 4. Male gender was associated with partial thrombus resolution. All other factors appear to not be statistically significant in impacting the development of a partial thrombus. This has been the first attempt to look at this new clinical entity.

Effect of Pre-Procedure Clopidogrel With Iliac Vein Stenting in Non-Thrombotic Vein Lesions.

OBJECTIVES: Iliac vein stenting is a relatively new procedure in the treatment of chronic venous insufficiency. Research has shown that it is a safe and effective form of treatment, however, one of the well-known risks is in-stent thrombosis. We hypothesize that a single 75 mg dose of Clopidogrel the night prior to the procedures along with a 3-month regimen post-op would decrease the 30-day thrombosis rate. METHODS: A retrospective study was performed on 3,518 patients from September 2012 to August 2018 who received an iliofemoral stent. Patients were broken down into 2 main groups: those given Clopidogrel post-stent and those given Clopidogrel both pre- and post-stent. In our practice, we prescribe a 3-month course of Clopidogrel after iliac vein stenting. Patients were also checked for any anticoagulant medications pre- and/or post-stent. The 30-day thrombosis rates were recorded for each patient. RESULTS: 1,205 patients received Clopidogrel pre-procedurally and post-procedurally, 1,941 patients received Clopidogrel only post-procedurally. 372 patients were excluded from the study because they were on other anti-coagulant medications. Mean follow-up for this cohort was 17 months. 112 total patients developed some degree of 30 day in-stent thrombosis (3.6%). 74 patients developed a complete thrombosis of the stent and 38 developed a partial (≤60% occlusion) thrombosis. Of the 1,205 patients who were on clopidogrel pre-stenting, 28 had a complete thrombosis and 10 had a partial in-stent thrombosis. Of the 1,941 patients on Clopidogrel only post-stenting, 46 had a complete thrombosis and 28 had a partial in-stent thrombosis. Using the Chi-squared test, there were no statistically significant differences between the group of patients receiving Clopidogrel pre- and post-stent vs. just post-stent with respect to 30-day any degree of thrombosis rates (complete and partial thrombosis) (p = .33). Using the Chi-squared test, there were no statistically significant differences between the group of patients receiving Clopidogrel pre- and post-stent vs. just post-stent with respect to 30-day complete thrombosis rates (p = .93). CONCLUSIONS: There appears to be no statistical difference in 30-day thrombosis rates between those receiving Clopidogrel the night prior vs. those who do not receive Clopidogrel the night prior. Therefore, we conclude that it is not necessary to give this single dose the night prior to iliac vein stenting procedures.

Extracellular histones, a new class of inhibitory molecules of CNS axonal regeneration.

Axonal regeneration in the mature CNS is limited by extracellular inhibitory factors. Triple knockout mice lacking the major myelin-associated inhibitors do not display spontaneous regeneration after injury, indicating the presence of other inhibitors. Searching for such inhibitors, we have detected elevated levels of histone H3 in human CSF 24 h after spinal cord injury. Following dorsal column lesions in mice and optic nerve crushes in rats, elevated levels of extracellular histone H3 were detected at the injury site. Similar to myelin-associated inhibitors, these extracellular histones induced growth cone collapse and inhibited neurite outgrowth. Histones mediate inhibition through the transcription factor Y-box-binding protein 1 and Toll-like receptor 2, and these effects are independent of the Nogo receptor. Histone-mediated inhibition can be reversed by the addition of activated protein C in vitro, and activated protein C treatment promotes axonal regeneration in the crushed optic nerve in vivo. These findings identify extracellular histones as a new class of nerve regeneration-inhibiting molecules within the injured CNS.

Dynamic balance between vesicle transport and microtubule growth enables neurite outgrowth.

Whole cell responses involve multiple subcellular processes (SCPs). To understand how balance between SCPs controls the dynamics of whole cell responses we studied neurite outgrowth in rat primary cortical neurons in culture. We used a combination of dynamical models and experiments to understand the conditions that permitted growth at a specified velocity and when aberrant growth could lead to the formation of dystrophic bulbs. We hypothesized that dystrophic bulb formation is due to quantitative imbalances between SCPs. Simulations predict redundancies between lower level sibling SCPs within each type of high level SCP. In contrast, higher level SCPs, such as vesicle transport and exocytosis or microtubule growth characteristic of each type need to be strictly coordinated with each other and imbalances result in stalling of neurite outgrowth. From these simulations, we predicted the effect of changing the activities of SCPs involved in vesicle exocytosis or microtubule growth could lead to formation of dystrophic bulbs. siRNA ablation experiments verified these predictions. We conclude that whole cell dynamics requires balance between the higher-level SCPs involved and imbalances can terminate whole cell responses such as neurite outgrowth.

A biomimetic gelatin-based platform elicits a pro-differentiation effect on podocytes through mechanotransduction.

Using a gelatin microbial transglutaminase (gelatin-mTG) cell culture platform tuned to exhibit stiffness spanning that of healthy and diseased glomeruli, we demonstrate that kidney podocytes show marked stiffness sensitivity. Podocyte-specific markers that are critical in the formation of the renal filtration barrier are found to be regulated in association with stiffness-mediated cellular behaviors. While podocytes typically de-differentiate in culture and show diminished physiological function in nephropathies characterized by altered tissue stiffness, we show that gelatin-mTG substrates with Young's modulus near that of healthy glomeruli elicit a pro-differentiation and maturation response in podocytes better than substrates either softer or stiffer. The pro-differentiation phenotype is characterized by upregulation of gene and protein expression associated with podocyte function, which is observed for podocytes cultured on gelatin-mTG gels of physiological stiffness independent of extracellular matrix coating type and density. Signaling pathways involved in stiffness-mediated podocyte behaviors are identified, revealing the interdependence of podocyte mechanotransduction and maintenance of their physiological function. This study also highlights the utility of the gelatin-mTG platform as an in vitro system with tunable stiffness over a range relevant for recapitulating mechanical properties of soft tissues, suggesting its potential impact on a wide range of research in cellular biophysics.