Salil Srivastava

Targeting the Nrf2–Keap1 antioxidant defence pathway for neurovascular protection in stroke

Abstract  Endogenous defence mechanisms by which the brain protects itself against noxious stimuli and recovers from ischaemic damage are a key target of stroke research. The loss of viable brain tissue in the ischaemic core region after stroke is associated with damage to the surrounding area known as the penumbra. Activation of the redox‐sensitive transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2) plays a pivotal role in the cellular defence against oxidative stress via transcriptional upregulation of phase II defence enzymes and antioxidant stress proteins. Although recent evidence implicates Nrf2 in neuroprotection, it is not known whether activation of this pathway within the neurovascular unit protects the brain against blood–brain barrier breakdown and cerebrovascular inflammation. Targeting the neurovascular unit should provide novel insights for effective treatment strategies and facilitate translation of experimental findings into clinical therapy. This review focuses on the cytoprotective role of Nrf2 in stroke and examines the evidence that the Nrf2–Keap1 defence pathway may serve as a therapeutic target for neurovascular protection.

Sulforaphane preconditioning of the Nrf2/HO-1 defense pathway protects the cerebral vasculature against blood-brain barrier disruption and neurological deficits in stroke

Disruption of the blood-brain barrier (BBB) and cerebral edema are the major pathogenic mechanisms leading to neurological dysfunction and death after ischemic stroke. The brain protects itself against infarction via activation of endogenous antioxidant defense mechanisms, and we here report the first evidence that sulforaphane-mediated preactivation of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target heme oxygenase-1 (HO-1) in the cerebral vasculature protects the brain against stroke. To induce ischemic stroke, Sprague-Dawley rats were subjected to 70 min middle cerebral artery occlusion (MCAo) followed by 4, 24, or 72 h reperfusion. Nrf2 and HO-1 protein expression was upregulated in cerebral microvessels of peri-infarct regions after 4-72 h, with HO-1 preferentially associated with perivascular astrocytes rather than the cerebrovascular endothelium. In naïve rats, treatment with sulforaphane increased Nrf2 expression in cerebral microvessels after 24h. Upregulation of Nrf2 by sulforaphane treatment prior to transient MCAo (1h) was associated with increased HO-1 expression in perivascular astrocytes in peri-infarct regions and cerebral endothelium in the infarct core. BBB disruption, lesion progression, as analyzed by MRI, and neurological deficits were reduced by sulforaphane pretreatment. As sulforaphane pretreatment led to a moderate increase in peroxynitrite generation, we suggest that hormetic preconditioning underlies sulforaphane-mediated protection against stroke. In conclusion, we propose that pharmacological or dietary interventions aimed to precondition the brain via activation of the Nrf2 defense pathway in the cerebral microvasculature provide a novel therapeutic approach for preventing BBB breakdown and neurological dysfunction in stroke.

TRPA1 is essential for the vascular response to environmental cold exposure

The cold-induced vascular response, consisting of vasoconstriction followed by vasodilatation, is critical for protecting the cutaneous tissues against cold injury. Whilst this physiological reflex response is historic knowledge, the mechanisms involved are unclear. Here by using a murine model of local environmental cold exposure, we show that TRPA1 acts as a primary vascular cold sensor, as determined through TRPA1 pharmacological antagonism or gene deletion. The initial cold-induced vasoconstriction is mediated via TRPA1-dependent superoxide production that stimulates α2C-adrenoceptors and Rho-kinase-mediated MLC phosphorylation, downstream of TRPA1 activation. The subsequent restorative blood flow component is also dependent on TRPA1 activation being mediated by sensory nerve-derived dilator neuropeptides CGRP and substance P, and also nNOS-derived NO. The results allow a new understanding of the importance of TRPA1 in cold exposure and provide impetus for further research into developing therapeutic agents aimed at the local protection of the skin in disease and adverse climates.

Cardioprotective effects of Cu((II))ATSM in human vascular smooth muscle cells and cardiomyocytes mediated by Nrf2 and DJ-1.

Cu(II)ATSM was developed as a hypoxia sensitive positron emission tomography agent. Recent reports have highlighted the neuroprotective properties of Cu(II)ATSM, yet there are no reports that it confers cardioprotection. We demonstrate that Cu(II)ATSM activates the redox-sensitive transcription factor Nrf2 in human coronary artery smooth muscle cells (HCASMC) and cardiac myocytes (HCM), leading to upregulation of antioxidant defense enzymes. Oral delivery of Cu(II)ATSM in mice induced expression of the Nrf2-regulated enzymes in the heart and aorta. In HCASMC, Cu(II)ATSM increased expression of the Nrf2 stabilizer DJ-1, and knockdown of Nrf2 or DJ-1 attenuated Cu(II)ATSM-mediated heme oxygenase-1 and NADPH quinone oxidoreductase-1 induction. Pre-treatment of HCASMC with Cu(II)ATSM protected against the pro-oxidant effects of angiotensin II (Ang II) by attenuating superoxide generation, apoptosis, proliferation and increases in intracellular calcium. Notably, Cu(II)ATSM-mediated protection against Ang II-induced HCASMC apoptosis was diminished by Nrf2 knockdown. Acute treatment with Cu(II)ATSM enhanced the association of DJ-1 with superoxide dismutase-1 (SOD1), paralleled by significant increases in intracellular Cu(II) levels and SOD1 activity. We describe a novel mechanism by which Cu(II)ATSM induces Nrf2-regulated antioxidant enzymes and protects against Ang II-mediated HCASMC dysfunction via activation of the Nrf2/DJ-1 axis. Cu(II)ATSM may provide a therapeutic strategy for cardioprotection via upregulation of antioxidant defenses.

Temporal and spatial distribution of Nrf2 in rat brain following stroke: quantification of nuclear to cytoplasmic Nrf2 content using a novel immunohistochemical technique

•  The redox‐sensitive transcription factor NF‐E2 related factor 2 (Nrf2) plays a key role in regulating adaptive cellular antioxidant defences, and activation of Nrf2 in stroke protects the brain against oxidative stress following ischaemia‐reperfusion injury. •  We report the first measurements of temporal and spatial distribution of Nrf2 in nuclear and cytoplasmic compartments in cells in the ischaemic core, peri‐infarct regions and contralateral hemisphere of rat brain following cerebral ischaemia‐reperfusion injury for 4, 24 or 72 h using a novel quantitative immunohistochemical technique, which was further validated in cultured bEnd.3 murine brain endothelial cells. •  Nrf2 expression in brain sections was increased in core and peri‐infarct regions after 24 h reperfusion, with levels remaining elevated only in peri‐infarct regions after 72 h. Pretreatment of rats with the Nrf2 inducer sulforaphane reduced core and peri‐infarct Nrf2 levels after 24 h reperfusion. •  The time course of stroke‐induced changes in nuclear to cytoplasmic Nrf2 content and its modulation by pretreatment with sulforaphane provide novel insights for targeting endogenous redox sensitive antioxidant pathways to ameliorate the damaging consequences of stroke.

A Novel α-Calcitonin Gene-Related Peptide Analogue Protects Against End-Organ Damage in Experimental Hypertension, Cardiac Hypertrophy and Heart Failure

Background: Research into the therapeutic potential of &agr;-calcitonin gene–related peptide (&agr;-CGRP) has been limited because of its peptide nature and short half-life. Here, we evaluate whether a novel potent and long-lasting (t½ ≥7 hours) acylated &agr;-CGRP analogue (&agr;Analogue) could alleviate and reverse cardiovascular disease in 2 distinct murine models of hypertension and heart failure in vivo. Methods: The ability of the &agr;Analogue to act selectively via the CGRP pathway was shown in skin by using a CGRP receptor antagonist. The effect of the &agr;Analogue on angiotensin II–induced hypertension was investigated over 14 days. Blood pressure was measured by radiotelemetry. The ability of the &agr;Analogue to modulate heart failure was studied in an abdominal aortic constriction model of murine cardiac hypertrophy and heart failure over 5 weeks. Extensive ex vivo analysis was performed via RNA analysis, Western blot, and histology. Results: The angiotensin II–induced hypertension was attenuated by cotreatment with the &agr;Analogue (50 nmol·kg–1·d–1, SC, at a dose selected for lack of long-term hypotensive effects at baseline). The &agr;Analogue protected against vascular, renal, and cardiac dysfunction, characterized by reduced hypertrophy and biomarkers of fibrosis, remodeling, inflammation, and oxidative stress. In a separate study, the &agr;Analogue reversed angiotensin II–induced hypertension and associated vascular and cardiac damage. The &agr;Analogue was effective over 5 weeks in a murine model of cardiac hypertrophy and heart failure. It preserved heart function, assessed by echocardiography, while protecting against adverse cardiac remodeling and apoptosis. Moreover, treatment with the &agr;Analogue was well tolerated with neither signs of desensitization nor behavioral changes. Conclusions: These findings, in 2 distinct models, provide the first evidence for the therapeutic potential of a stabilized &agr;Analogue, by mediating (1) antihypertensive effects, (2) attenuating cardiac remodeling, and (3) increasing angiogenesis and cell survival to protect against and limit damage associated with the progression of cardiovascular diseases. This indicates the therapeutic potential of the CGRP pathway and the possibility that this injectable CGRP analogue may be effective in cardiac disease.

The anti-ageing hormone klotho induces Nrf2-mediated antioxidant defences in human aortic smooth muscle cells

Vascular ageing in conditions such as atherosclerosis, diabetes and chronic kidney disease, is associated with the activation of the renin angiotensin system (RAS) and diminished expression of antioxidant defences mediated by the transcription factor nuclear factor erythroid 2‐related factor 2 (Nrf2). The anti‐ageing hormone klotho promotes longevity and protects against cardiovascular and renal diseases. Klotho has been shown to activate Nrf2 and attenuate oxidative damage in neuronal cells, however, the mechanisms by which it protects against vascular smooth muscle cell VSMC dysfunction elicited by Angiotensin II (AngII) remain to be elucidated. AngII contributes to vascular ageing and atherogenesis by enhancing VSMC oxidative stress, senescence and apoptosis. This study demonstrates that soluble klotho (1 nM, 24 hrs) significantly induces expression of Nrf2 and the antioxidant enzymes haeme oxygenase (HO‐1) and peroxiredoxin‐1 (Prx‐1) and enhances glutathione levels in human aortic smooth muscle cells (HASMC). Silencing of Nrf2 attenuated the induction of HO‐1 and Prx‐1 expression by soluble klotho. Furthermore, soluble klotho protected against AngII‐mediated HASMC apoptosis and senescence via activation of Nrf2. Thus, our findings highlight a novel Nrf2‐mediated mechanism underlying the protective actions of soluble klotho in HAMSC. Targeting klotho may thus represent a therapeutic strategy against VSMC dysfunction and cardiovascular ageing.

Transient receptor potential canonical 5 (TRPC5) protects against pain and vascular inflammation in arthritis and joint inflammation

Objective Transient receptor potential canonical 5 (TRPC5) is functionally expressed on a range of cells including fibroblast-like synoviocytes, which play an important role in arthritis. A role for TRPC5 in inflammation has not been previously shown in vivo. We investigated the contribution of TRPC5 in arthritis. Methods Male wild-type and TRPC5 knockout (KO) mice were used in a complete Freunds adjuvant (CFA)-induced unilateral arthritis model, assessed over 14 days. Arthritis was determined by measurement of knee joint diameter, hindlimb weightbearing asymmetry and pain behaviour. Separate studies involved chronic pharmacological antagonism of TRPC5 channels. Synovium from human postmortem control and inflammatory arthritis samples were investigated for TRPC5 gene expression. Results At baseline, no differences were observed. CFA-induced arthritis resulted in increased synovitis in TRPC5 KO mice assessed by histology. Additionally, TRPC5 KO mice demonstrated reduced ispilateral weightbearing and nociceptive thresholds (thermal and mechanical) following CFA-induced arthritis. This was associated with increased mRNA expression of inflammatory mediators in the ipsilateral synovium and increased concentration of cytokines in synovial lavage fluid. Chronic treatment with ML204, a TRPC5 antagonist, augmented weightbearing asymmetry, secondary hyperalgesia and cytokine concentrations in the synovial lavage fluid. Synovia from human inflammatory arthritis demonstrated a reduction in TRPC5 mRNA expression. Conclusions Genetic deletion or pharmacological blockade of TRPC5 results in an enhancement in joint inflammation and hyperalgesia. Our results suggest that activation of TRPC5 may be associated with an endogenous anti-inflammatory/analgesic pathway in inflammatory joint conditions.

Phosphatidylinositol-3,4,5-trisphosphate stimulates Ca2+ elevation and Akt phosphorylation to constitute a major mechanism of thromboxane A2 formation in human platelets

Phosphatidylinositol trisphosphate (PIP3) has been implicated in many platelet functions however many of the mechanisms need clarification. We have used cell permeable analogues of PIP3,1-O-(1,2-di-palmitoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC16-PIP3) or 1-O-(1,2-di-octanoyl-sn-glyero-3-O-phosphoryl)-D-myo-inositol-3,4,5-trisphosphate (DiC8-PIP3) to study their effects on activation on washed human platelets. Addition of either DiC8- or DiC16-PIP3 to human platelets induced aggregation in the presence of extracellular Ca(2+). This was reduced by the presence of indomethacin, the phospholipase C inhibitor U73122 and apyrase. DiC8-PIP3 induced the phosphorylation of Akt-Ser(473) which was reduced by the Akt inhibitor IV, wortmannin and EGTA (suggesting a dependence on Ca(2+) entry). In Fura2 loaded platelets DiC8-PIP3 was effective at increasing intracellular Ca(2+) in a distinct and transient manner that was reduced in the presence of indomethacin, U73122 and 2-aminoethyl diphenylborinate (2APB). Ca(2+) elevation was reduced by the non-SOCE inhibitor LOE908 and also by the SOCE inhibitor BTP2. DiC8-PIP3 induced the release of Ca(2+) from stores which was not affected by the proton dissipating agent bafilomycin A1 and was more potent than the two-pore channel agonist DiC8-PI[3,5]P2 suggesting release from an endoplasmic reticulum type store. DiC8-PIP3 weakly induced the tyrosine phosphorylation of Syk but not of PLCγ2. Finally like thrombin DiC8-PIP3 induced the formation of thromboxane B2 that was inhibited by the Akt inhibitor IV. These studies suggest that PIP3 via Ca(2+) elevation and Akt phosphorylation forms a central role in thromboxane A2 formation and the amplification of platelet activation.

Isolation, Culture, and Characterization of Vascular Smooth Muscle Cells.

Smooth muscle cells (SMC) are the predominant cell type involved in the pathogenesis of atherosclerosis, vascular calcification and restenosis after angioplasty; however, they are also important in the de novo formation of blood vessels through differentiation of mesenchymal cells under the influence of mediators secreted by endothelial cells. In angiogenesis, vascular SMC are formed by proliferation of existing SMC or maturation and differntiation of pericytes. Experimental findings have demonstrated a potential role of putative smooth muscle progenitor cells in the circulation or within adult tissues and the perivascular adventitia in the development of atherosclerotic plaques, restenosis and angiogenesis. Modulation of vascular smooth muscle phenotype, SMC migration and hypertrophy are now recognized as key events in the development of vascular diseases. This has led to an increase in experimental research on SMC function in response to growth factors, extracellular matrix components, modified lipoproteins, biomechanical forces and other pro-atherogenic and pro-angiogenic mediators to address the cellular mechanisms involved. This chapter highlights well established methodologies used for vascular SMC and pericyte isolation and culture as well as their characterisation. A better understanding of vascular SMC and pericyte biology and their phenotypic modulation is required to identify therapeutic strategies to target angiogenesis and treat cardiovascular diseases.