investigating-ischemic-stroke-recurrent-injury-secondary-middle

Investigating Ischemic Stroke Recurrent Injury in a Secondary Middle Cerebral Artery Occlusion Model in Mouse

Stroke is the second leading global cause of mortality and disability, with recurrent strokes contributing significantly to poor outcomes and increased ...

University of California, Davis

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3(+) memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3 high TEM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3(+) lymphocytes/mm(2) of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3(+) T cells and HLA-DR(+) T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis.

Kv1.3 in psoriatic disease: PAP-1, a small molecule inhibitor of Kv1.3 is effective in the SCID mouse psoriasis--xenograft model.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation-contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models.

Inhibitors of the voltage-gated K channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up-regulated on "M1-like" microglia, we here tested the therapeutic hypothesis that the brain-penetrant small-molecule Kv1.3-inhibitor PAP-1 reduces secondary inflammatory damage after ischemia/reperfusion.

Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke.

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.

Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4-mediated calcium entry in microglia.

Myocardial infarction (MI) and osteoporotic fracture are leading causes of morbidity and mortality, and epidemiological evidence linking their incidence suggests possible crosstalk. MI can exacerbate atherosclerosis through the sympathetic nervous system (SNS) activation and β adrenoreceptor-mediated release of hematopoietic stem cells, leading to monocytosis. We hypothesized that this same pathway initiates systemic bone loss following MI, since osteoclasts differentiate from monocytes. In this study, MI was created with left anterior descending artery ligation in 12-week-old male mice (n = 24) randomized to β -adrenergic receptor (AR) antagonist (SR 59230A) treatment or no treatment for 10 days postoperatively. Additional mice (n = 21, treated and untreated) served as unoperated controls. Bone mineral density (BMD), bone mineral content (BMC), and body composition were quantified at baseline and 10 days post-MI using dual-energy x-ray absorptiometry; circulating monocyte levels were quantified and the L5 vertebral body and femur were analyzed with microcomputed tomography 10 days post-MI. We found that MI led to circulating monocyte levels increases, BMD and BMC decreases at the femur and lumbar spine in MI mice (-6.9% femur BMD, -3.5% lumbar BMD), and trabecular bone volume decreases in MI mice compared with control mice. β -AR antagonist treatment appeared to diminish the bone loss response (-5.3% femur BMD, -1.2% lumbar BMD), though these results were somewhat inconsistent. Clinical significance: These results suggest that MI leads to systemic bone loss, but that the SNS may not be a primary modulator of this response; bone loss and increased fracture risk may be important clinical comorbidities following MI or other ischemic injuries.

Systemic bone loss following myocardial infarction in mice.

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC = 1 μM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel Na1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC = 60 nM) and SKA-378 is the most potent inhibitor of Na1.6 (IC = 28 μM) compared to Na1.2 (IC = 118 μM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy.

The voltage-gated sodium Na1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human Na1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human Na1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with ICs of 7 nM and 4 nM for hNa1.7 and more than 1000-fold selectivity over human Na1.1, Na1.3, Na1.4, Na1.5, Na1.8, and Na1.9 channels. PTx2-3127 inhibits Na1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human Na1.7 channels have transformative potential to define a new class of biologics to treat pain.

Computational design of peptides to target Na1.7 channel with high potency and selectivity for the treatment of pain.

 Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality with an estimated 80% of SHS-related deaths attributed to cardiovascular causes. Public health measures and smoking bans have been successful both in reducing SHS exposure and improving cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a promising target for mitigating SHS exposure-induced cardiovascular outcomes.  The objectives of this study were to determine 1) effects of environmentally relevant SHS exposure on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of an sEH inhibitor (TPPU) can reduce the adverse cardiovascular effects of SHS exposure.  Male C57BL/6J mice (11 weeks old) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to filtered air or 3 mg/m of SHS (6 hr/d, 5 d/wk) for 12 weeks, followed by 4 weeks of recovery in filtered air. Some mice received TPPU in drinking water (15 mg/L) throughout SHS exposure. BP, heart rate (HR), HR variability (HRV), baroreflex sensitivity (BRS), and BP variability were determined monthly.  SHS exposure significantly decreased 1) short-term HRV by ∼20% ( < 0.05) within 4 weeks; 2) overall HRV with maximum effect at 12 weeks (-15%,  < 0.05); 3) pulse pressure (-8%,  < 0.05) as early as week 4; and 4) BRS with maximum effect at 12 weeks (-11%,  < 0.05). Four weeks of recovery following 12 weeks of SHS ameliorated all SHS-induced cardiovascular detriments. Importantly, mice exposed to TPPU in drinking water during SHS-related exposure were protected from SHS cardiovascular consequences.  The data suggest that 1) environmental relevant SHS exposure significantly alters cardiac autonomic function and BP regulation; 2) cardiovascular consequences from SHS can be reversed by discontinuing SHS exposure; and 3) inhibiting sEH can prevent SHS-induced cardiovascular consequences.

Effects of chronic secondhand smoke exposure on cardiovascular regulation and the role of soluble epoxide hydrolase in mice.

Myocardial infarction (MI) and osteoporotic fracture are two of the leading causes of morbidity and mortality worldwide. We have previously established that MI in mice directly causes post-traumatic systemic bone loss and that the sympathetic nervous system plays a role in this response. However, the systemic injury response is mediated by multiple systems. In this study, we investigated the role of complement 5a (C5a), one of the main mediators driving multiple organ dysfunction after trauma. MI was surgically induced in 12-week-old male C57BL/6 J mice (B6/J), C5a receptor 1 knockout mice (C5aR1) and B10·D2-Hc0 H2d H2-T18c/oSnJ (B10·D2), (JAX Strain # 000461) mice that are deficient in serum C5. Systemic and localized bone changes were analyzed at 7-, 14- and 28-days post-MI using micro-computed tomography and three-point bending mechanical testing. Osteoclast number and activity was quantified using tartrate-resistant acid phosphatase (TRAP) staining, and voluntary activity levels were measured using open field. We found that MI induced peak trabecular bone loss 7 days after injury in the L5 vertebral body and caused reductions in femoral cortical bone 28 days post-MI. However, MI did not impact femoral trabecular bone in this timeframe. B10·D2 mice had reduced trabecular and cortical bone morphology compared to B6 and C5aR1 mice, but did not exhibit an altered response to MI. Osteoclast activity 7-days post-MI was increased in C5aR1 mice compared to B6 and B10·D2 mice, but MI did not impact osteoclast activity at this time point. Altogether, these findings suggest that C5a may influence overall response to MI and bone morphology instead of post-traumatic systemic bone loss response following MI, though likely not as a primary mechanism.

Article	Year
Kv1.3 in psoriatic disease: PAP-1, a small molecule inhibitor of Kv1.3 is effective in the SCID mouse psoriasis--xenograft model. Journal of autoimmunity\| PubMed ID: 25175978	2014
In Vivo Cannulation Methods for Cardiomyocytes Isolation from Heart Disease Models. PloS one\| PubMed ID: 27500929	2016
Inhibition of the potassium channel Kv1.3 reduces infarction and inflammation in ischemic stroke. Annals of clinical and translational neurology\| PubMed ID: 29468176	2018
Biophysical basis for Kv1.3 regulation of membrane potential changes induced by P2X4-mediated calcium entry in microglia. Glia\| PubMed ID: 32525239	2020
Systemic bone loss following myocardial infarction in mice. Journal of orthopaedic research : official publication of the Orthopaedic Research Society\| PubMed ID: 32965732	2021
Riluzole and novel naphthalenyl substituted aminothiazole derivatives prevent acute neural excitotoxic injury in a rat model of temporal lobe epilepsy. Neuropharmacology\| PubMed ID: 36436594	2023
Computational design of peptides to target Na1.7 channel with high potency and selectivity for the treatment of pain. eLife\| PubMed ID: 36576241	2022
Effects of chronic secondhand smoke exposure on cardiovascular regulation and the role of soluble epoxide hydrolase in mice. Frontiers in physiology\| PubMed ID: 37362438	2023
Investigating the role of complement 5a in systemic bone loss after myocardial infarction. Bone\| PubMed ID: 40412474	2025

Yi-Je Chen

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