Characterization of CPH:SA microparticle-based delivery of interleukin-1 alpha for cancer immunotherapy.

Background: Interleukin-1 alpha (IL-1α) is a pro-inflammatory cytokine that can activate immune effector cells and trigger anti-tumor immune responses. However, dose-limiting toxicities including cytokine storm and hypotension has limited its use in the clinic as a cancer therapy. We propose that polymeric microparticle (MP)-based delivery of IL-1α will suppress the acute pro-inflammatory side effects by allowing for slow and controlled release of IL-1α systemically, while simultaneously triggering an anti-tumor immune response. Methods: Polyanhydride copolymers composed of 1,6-bis-(p-carboxyphenoxy)-hexane:sebacic 20:80 (CPH:SA 20:80) was utilized to fabricate MPs. Recombinant IL-1α (rIL-1α) was encapsulated into CPH:SA 20:80 MPs (IL-1α-MPs) and the MPs were characterized by size, charge, loading efficiency, and in-vitro release and activity of IL-1α. IL-1α-MPs were injected intraperitonially into head and neck squamous cell carcinoma (HNSCC)-bearing C57Bl/6 mice and monitored for changes in weight, tumor growth, circulating cytokines/chemokines, hepatic and kidney enzymes, blood pressure, heart rate, and tumor-infiltrating immune cells. Results: CPH:SA IL-1α-MPs demonstrated sustained release kinetics of IL-1α (100% protein released over 8-10 days) accompanied by minimal weight loss and systemic inflammation compared to rIL-1α-treated mice. Blood pressure measured by radiotelemetry in conscious mice demonstrates that rIL-1α-induced hypotension was prevented in IL-1α-MP-treated mice. Liver and kidney enzymes were within normal range for all control and cytokine-treated mice. Both rIL-1α and IL-1α-MP-treated mice showed similar delays in tumor growth and similar increases in tumor-infiltrating CD3+ T cells, macrophages, and dendritic cells. Conclusions: CPH:SA-based IL-1α-MPs generated a slow and sustained systemic release of IL-1α resulting in reduced weight loss, systemic inflammation, and hypotension accompanied by an adequate anti-tumor immune response in HNSCC-tumor bearing mice. Therefore, MPs based on CPH:SA formulations may be promising as delivery vehicles for IL-1α to achieve safe, effective, and durable antitumor responses for HNSCC patients.

Plasticity of cerebral microvascular structure and mechanics during hypertension and following recovery of arterial pressure.

Changes in vascular structure contribute to vascular events and loss of brain health. We examined changes in cerebral arterioles at the onset of hypertension and the hypothesis that alterations during hypertension would recover with the return of mean arterial pressure (MAP) to normal. MAP was measured with radiotelemetry in awake male C57BL/6J mice at baseline and during infusion of vehicle or angiotensin II (ANG II, 1.4 mg/kg/day using osmotic pumps) for 28 days, followed by a 28-day recovery. With ANG II treatment, MAP increased through day 28. On day 30, MAP began to recover, reaching levels not different from vehicle on day 37. We measured intravascular pressure, diameter, wall thickness (WT), wall:lumen ratio (W:L), cross-sectional area (CSA), and slope of the tangential elastic modulus (ET) in maximally dilated arterioles. Variables were similar in both groups at day 1, with no significant change with vehicle treatment. With ANG II treatment, CSA, WT, and W:L increased on days 7-28. Internal and external diameter was reduced at 14 and 28 days. ET versus wall stress was reduced on days 7-28. During recovery, the diameter remained at days 14 and 28 values, whereas other variables returned partly or completely to normal. Thus, CSA, WT, W:L, and ET versus wall stress changed rapidly during hypertension and recovered with MAP. In contrast, inward remodeling developed slowly and did not recover. This lack of recovery has mechanistic implications for the long-term impact of hypertension on vascular determinants of brain health.NEW & NOTEWORTHY Changes in vascular structure contribute to vascular events and loss of brain health. We examined the inherent structural plasticity of cerebral arterioles during and after a period of hypertension. Arteriolar wall thickness, diameter, wall-to-lumen ratio, and biological stiffness changed rapidly during hypertension and recovered with blood pressure. In contrast, inward remodeling developed slowly and did not recover. This lack of recovery of arteriolar diameter has implications for the long-term impact of hypertension on vascular determinants of brain health.

Increased receptor activity-modifying protein 1 in the nervous system is sufficient to protect against autonomic dysregulation and hypertension.

Calcitonin gene-related peptide (CGRP) can cause migraines, yet it is also a potent vasodilator that protects against hypertension. Given the emerging role of CGRP-targeted antibodies for migraine prevention, an important question is whether the protective actions of CGRP are mediated by vascular or neural CGRP receptors. To address this, we have characterized the cardiovascular phenotype of transgenic nestin/hRAMP1 mice that have selective elevation of a CGRP receptor subunit in the nervous system, human receptor activity-modifying protein 1 (hRAMP1). Nestin/hRAMP1 mice had relatively little hRAMP1 RNA in blood vessels and intravenous injection of CGRP caused a similar blood pressure decrease in transgenic and control mice. At baseline, nestin/hRAMP1 mice exhibited similar mean arterial pressure, heart rate, baroreflex sensitivity, and sympathetic vasomotor tone as control mice. We previously reported that expression of hRAMP1 in all tissues favorably improved autonomic regulation and attenuated hypertension induced by angiotensin II (Ang II). Similarly, in nestin/hRAMP1 mice, hypertension caused by Ang II or phenylephrine was greatly attenuated, and associated autonomic dysregulation and increased sympathetic vasomotor tone were diminished or abolished. We conclude that increased expression of neuronal CGRP receptors is sufficient to induce a protective change in cardiovascular autonomic regulation with implications for migraine therapy.

Exercise prevents development of autonomic dysregulation and hyperalgesia in a mouse model of chronic muscle pain.

Chronic musculoskeletal pain (CMP) conditions, like fibromyalgia, are associated with widespread pain and alterations in autonomic functions. Regular physical activity prevents the development of CMP and can reduce autonomic dysfunction. We tested if there were alterations in autonomic function of sedentary mice with CMP, and whether exercise reduced the autonomic dysfunction and pain induced by CMP. Chronic musculoskeletal pain was induced by 2 intramuscular injections of pH 5.0 in combination with a single fatiguing exercise task. A running wheel was placed into cages so that the mouse had free access to it for either 5 days or 8 weeks (exercise groups) and these animals were compared to sedentary mice without running wheels. Autonomic function and nociceptive withdrawal thresholds of the paw and muscle were assessed before and after induction of CMP in exercised and sedentary mice. In sedentary mice, we show decreased baroreflex sensitivity, increased blood pressure variability, decreased heart rate variability, and decreased withdrawal thresholds of the paw and muscle 24 hours after induction of CMP. There were no sex differences after induction of the CMP in any outcome measure. We further show that both 5 days and 8 weeks of physical activity prevent the development of autonomic dysfunction and decreases in withdrawal threshold induced by CMP. Thus, this study uniquely shows the development of autonomic dysfunction in animals with chronic muscle hyperalgesia, which can be prevented with as little as 5 days of physical activity, and suggest that physical activity may prevent the development of pain and autonomic dysfunction in people with CMP.

Angiotensin-dependent autonomic dysregulation precedes dilated cardiomyopathy in a mouse model of muscular dystrophy.

NEW FINDINGS: What is the central question of this study? Is autonomic dysregulation in a mouse model of muscular dystrophy dependent on left ventricular systolic dysfunction and/or activation of the renin-angiotensin system (RAS) and does it predict development of dilated cardiomyopathy (DCM)? What is the main finding and its importance? The results demonstrate that autonomic dysregulation precedes and predicts left ventricular dysfunction and DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice. The autonomic dysregulation is prevented by treatment of young Sgcd-/- mice with the angiotensin II type 1 receptor blocker losartan. Measurements of RAS activation and autonomic dysregulation may predict risk of DCM, and therapies targeting the RAS and autonomic dysregulation at a young age may slow disease progression in patients. Sarcoglycan mutations cause muscular dystrophy. Patients with muscular dystrophy develop autonomic dysregulation and dilated cardiomyopathy (DCM), but the temporal relationship and mechanism of autonomic dysregulation are not well understood. We hypothesized that activation of the renin-angiotensin system (RAS) causes autonomic dysregulation prior to development of DCM in sarcoglycan-δ-deficient (Sgcd-/-) mice and that the severity of autonomic dysfunction at a young age predicts the severity of DCM at older ages. At 10-12 weeks of age, when left ventricular function assessed by echocardiography remained normal, Sgcd-/- mice exhibited decreases in arterial pressure, locomotor activity, baroreflex sensitivity and cardiovagal tone and increased sympathetic tone compared with age-matched C57BL/6 control mice (P < 0.05). Systemic and skeletal muscle RAS were activated, and angiotensin II type 1 receptor (AT1 R) expression, superoxide and fibrosis were increased in dystrophic skeletal muscle (P < 0.05). Treatment with the AT1 R blocker losartan for 7-9 weeks beginning at 3 weeks of age prevented or strongly attenuated the abnormalities in Sgcd-/- mice (P < 0.05). Repeated assessment of phenotypes between 10 and 75 weeks of age demonstrated worsening of autonomic function, progressive cardiac dysfunction and DCM and increased mortality in Sgcd-/- mice. High sympathetic tone predicted subsequent left ventricular dysfunction. We conclude that activation of the RAS causes severe autonomic dysregulation in young Sgcd-/- mice, which portends a worse long-term prognosis. Therapeutic targeting of the RAS at a young age may improve autonomic function and slow disease progression in muscular dystrophy.

The link between stress disorders and autonomic dysfunction in muscular dystrophy.

Muscular dystrophy is a progressive disease of muscle weakness, muscle atrophy and cardiac dysfunction. Patients afflicted with muscular dystrophy exhibit autonomic dysfunction along with cognitive impairment, severe depression, sadness, and anxiety. Although the psychological aspects of cardiovascular disorders and stress disorders are well known, the physiological mechanism underlying this relationship is not well understood, particularly in muscular dystrophy. Therefore, the goal of this perspective is to highlight the importance of autonomic dysfunction and psychological stress disorders in the pathogenesis of muscular dystrophy. This article will for the first time-(i) outline autonomic mechanisms that are common to both psychological stress and cardiovascular disorders in muscular dystrophy; (ii) propose therapies that would improve behavioral and autonomic functions in muscular dystrophy.

Chronic oral administration of Ang-(1-7) improves skeletal muscle, autonomic and locomotor phenotypes in muscular dystrophy.

Muscular dystrophies are a group of heterogeneous genetic disorders that cause progressive muscle weakness and wasting, dilated cardiomyopathy and early mortality. There are different types of muscular dystrophies with varying aetiologies but they all have a common hallmark of myofibre degeneration, atrophy and decreased mobility. Mutation in Sgcd (sarcoglycan-δ), a subunit of dystrophin glycoprotein complex, causes LGMD2F (limb girdle muscular dystrophy 2F). Previously, we have reported that Sgcd-deficient (Sgcd-/-) mice exhibit AngII (angiotensin II)-induced autonomic and skeletal muscle dysfunction at a young age, which contributes to onset of dilated cardiomyopathy and mortality at older ages. Two counter-regulatory RAS (renin-angiotensin system) pathways have been identified: deleterious actions of AngII acting on the AT1R (AngII type 1 receptor) compared with the protective actions of Ang-(1-7) [angiotensin-(1-7)] acting on the receptor Mas. We propose that the balance between the AngII/AT1R and Ang-(1-7)/Mas axes is disturbed in Sgcd-/- mice. Control C57BL/6J and Sgcd-/- mice were treated with Ang-(1-7) included in hydroxypropyl ß-cyclodextrin (in drinking water) for 8-9 weeks beginning at 3 weeks of age. Ang-(1-7) treatment restored the AngII/AT1R compared with Ang-(1-7)/Mas balance, decreased oxidative stress and fibrosis in skeletal muscle, increased locomotor activity, and prevented autonomic dysfunction without lowering blood pressure in Sgcd-/- mice. Our results suggest that correcting the early autonomic dysregulation by administering Ang-(1-7) or enhancing its endogenous production may provide a novel therapeutic approach in muscular dystrophy.

Autonomic, locomotor and cardiac abnormalities in a mouse model of muscular dystrophy: targeting the renin-angiotensin system.

New Findings What is the topic of this review? This symposium report summarizes autonomic, cardiac and skeletal muscle abnormalities in sarcoglycan-δ-deficient mice (Sgcd-/-), a mouse model of limb girdle muscular dystrophy, with emphasis on the roles of autonomic dysregulation and activation of the renin-angiotensin system at a young age. What advances does it highlight? The contributions of the autonomic nervous system and the renin-angiotensin system to the pathogenesis of muscular dystrophy are highlighted. Results demonstrate that autonomic dysregulation precedes and predicts later development of cardiac dysfunction in Sgcd-/- mice and that treatment of young Sgcd-/- mice with the angiotensin type 1 receptor antagonist losartan or with angiotensin-(1-7) abrogates the autonomic dysregulation, attenuates skeletal muscle pathology and increases spontaneous locomotor activity. Muscular dystrophies are a heterogeneous group of genetic muscle diseases characterized by muscle weakness and atrophy. Mutations in sarcoglycans and other subunits of the dystrophin-glycoprotein complex cause muscular dystrophy and dilated cardiomyopathy in animals and humans. Aberrant autonomic signalling is recognized in a variety of neuromuscular disorders. We hypothesized that activation of the renin-angiotensin system contributes to skeletal muscle and autonomic dysfunction in mice deficient in the sarcoglycan-δ (Sgcd) gene at a young age and that this early autonomic dysfunction contributes to the later development of left ventricular (LV) dysfunction and increased mortality. We demonstrated that young Sgcd-/- mice exhibit histopathological features of skeletal muscle dystrophy, decreased locomotor activity and severe autonomic dysregulation, but normal LV function. Autonomic regulation continued to deteriorate in Sgcd-/- mice with age and was accompanied by LV dysfunction and dilated cardiomyopathy at older ages. Autonomic dysregulation at a young age predicted later development of LV dysfunction and higher mortality in Sgcd-/- mice. Treatment of Sgcd-/- mice with the angiotensin type 1 receptor blocker losartan for 8-9 weeks, beginning at 3 weeks of age, decreased fibrosis and oxidative stress in skeletal muscle, increased locomotor activity and prevented autonomic dysfunction. Chronic infusion of the counter-regulatory peptide angiotensin-(1-7) resulted in similar protection. We conclude that activation of the renin-angiotensin system, at a young age, contributes to skeletal muscle and autonomic dysfunction in muscular dystrophy. We speculate that the latter is mediated via abnormal sensory nerve and/or cytokine signalling from dystrophic skeletal muscle to the brain and contributes to age-related LV dysfunction, dilated cardiomyopathy, arrhythmias and premature death. Therefore, correcting the early autonomic dysregulation and renin-angiotensin system activation may provide a novel therapeutic approach in muscular dystrophy.

Regulator of G protein signaling 2 deficiency causes endothelial dysfunction and impaired endothelium-derived hyperpolarizing factor-mediated relaxation by dysregulating Gi/o signaling.

Regulator of G protein signaling 2 (RGS2) is a GTPase-activating protein for G(q/11)α and G(i/o)α subunits. RGS2 deficiency is linked to hypertension in mice and humans, although causative mechanisms are not understood. Because endothelial dysfunction and increased peripheral resistance are hallmarks of hypertension, determining whether RGS2 regulates microvascular reactivity may reveal mechanisms relevant to cardiovascular disease. Here we have determined the effects of systemic versus endothelium- or vascular smooth muscle-specific deletion of RGS2 on microvascular contraction and relaxation. Contraction and relaxation of mesenteric resistance arteries were analyzed in response to phenylephrine, sodium nitroprusside, or acetylcholine with or without inhibitors of nitric oxide (NO) synthase or K(+) channels that mediate endothelium-derived hyperpolarizing factor (EDHF)-dependent relaxation. The results showed that deleting RGS2 in vascular smooth muscle had minor effects. Systemic or endothelium-specific deletion of RGS2 strikingly inhibited acetylcholine-evoked relaxation. Endothelium-specific deletion of RGS2 had little effect on NO-dependent relaxation but markedly impaired EDHF-dependent relaxation. Acute, inducible deletion of RGS2 in endothelium did not affect blood pressure significantly. Impaired EDHF-mediated vasodilatation was rescued by blocking G(i/o)α activation with pertussis toxin. These findings indicated that systemic or endothelium-specific RGS2 deficiency causes endothelial dysfunction resulting in impaired EDHF-dependent vasodilatation. RGS2 deficiency enables endothelial G(i/o) activity to inhibit EDHF-dependent relaxation, whereas RGS2 sufficiency facilitates EDHF-evoked relaxation by squelching endothelial G(i/o) activity. Mutation or down-regulation of RGS2 in hypertension patients therefore may contribute to endothelial dysfunction and defective EDHF-dependent relaxation. Blunting G(i/o) signaling might improve endothelial function in such patients.

Cold-impaired cardiac performance in rats is only partially overcome by cold acclimation.

The consequences of acute hypothermia include impaired cardiovascular performance, ultimately leading to circulatory collapse. We examined the extent to which this results from intrinsic limitations to cardiac performance or physiological dysregulation/autonomic imbalance, and whether chronic cold exposure could ameliorate the impaired function. Wistar rats were held at a 12 h:12 h light:dark (L:D) photoperiod and room temperature (21°C; euthermic controls), or exposed to a simulated onset of winter in an environmental chamber by progressive acclimation to 1 h:23 h L:D and 4°C over 4 weeks. In vivo, acute cold exposure (core temperature, T(b)=25°C) resulted in hypotension (approximately -20%) due to low cardiac output (approximately -30%) accompanying a bradycardia (approximately -50%). Cold acclimation (CA) induced only partial compensation for this challenge, including increased coronary flow at T(b)=37°C (but not at T(b)=25°C), maintenance of ventricular capillarity and altered sympathovagal balance (increased low:high frequency in power spectral analysis, PSA), suggesting physiological responses alone were insufficient to maintain cardiovascular performance. However, PSA showed maintenance of cardiorespiratory coupling on acute cold exposure in both groups. Ex vivo cardiac performance revealed no change in intrinsic heart rate, but a mechanical impairment of cardiac function at low temperatures following CA. While CA involved an increased capacity for ß-oxidation, there was a paradoxical reduction in developed pressure as a result of adrenergic down-regulation. These data suggest that integrated plasticity is the key to cardiovascular accommodation of chronic exposure to a cold environment, but with the potential for improvement by intervention, for example with agents such as non-catecholamine inotropes.

Methods of assessing vagus nerve activity and reflexes.

The methods used to assess cardiac parasympathetic (cardiovagal) activity and its effects on the heart in both humans and animal models are reviewed. Heart rate (HR)-based methods include measurements of the HR response to blockade of muscarinic cholinergic receptors (parasympathetic tone), beat-to-beat HR variability (HRV) (parasympathetic modulation), rate of post-exercise HR recovery (parasympathetic reactivation), and reflex-mediated changes in HR evoked by activation or inhibition of sensory (afferent) nerves. Sources of excitatory afferent input that increase cardiovagal activity and decrease HR include baroreceptors, chemoreceptors, trigeminal receptors, and subsets of cardiopulmonary receptors with vagal afferents. Sources of inhibitory afferent input include pulmonary stretch receptors with vagal afferents and subsets of visceral and somatic receptors with spinal afferents. The different methods used to assess cardiovagal control of the heart engage different mechanisms, and therefore provide unique and complementary insights into underlying physiology and pathophysiology. In addition, techniques for direct recording of cardiovagal nerve activity in animals; the use of decerebrate and in vitro preparations that avoid confounding effects of anesthesia; cardiovagal control of cardiac conduction, contractility, and refractoriness; and noncholinergic mechanisms are described. Advantages and limitations of the various methods are addressed, and future directions are proposed.

Receptor activity-modifying protein 1 increases baroreflex sensitivity and attenuates Angiotensin-induced hypertension.

Calcitonin gene-related peptide (CGRP) is a powerful vasodilator that interacts with the autonomic nervous system. A subunit of the CGRP receptor complex, receptor activity-modifying protein 1 (RAMP1), is required for trafficking of the receptor to the cell surface and high-affinity binding to CGRP. We hypothesized that upregulation of RAMP1 would favorably enhance autonomic regulation and attenuate hypertension. Blood pressure, heart rate, and locomotor activity were measured by radiotelemetry in transgenic mice with ubiquitous expression of human RAMP1 (hRAMP1) and littermate controls. Compared with control mice, hRAMP1 mice exhibited similar mean arterial pressure, a lower mean heart rate, increased heart rate variability, reduced blood pressure variability, and increased baroreflex sensitivity (2.83+/-0.20 versus 1.49+/-0.10 ms/mm Hg in controls; P<0.05). In control mice, infusion of angiotensin II (Ang-II) increased mean arterial pressure from 118+/-2 mm Hg to 153+/-4 and 174+/-6 mm Hg after 7 and 14 days of infusion, respectively (P<0.05). In contrast, Ang-II hypertension was markedly attenuated in hRAMP1 mice with corresponding values of mean arterial pressure of 111+/-2, 119+/-2, and 132+/-3 mm Hg. Ang-II induced decreases in baroreflex sensitivity and heart rate variability, and increases in blood pressure variability observed in control mice were also abrogated or reversed in hRAMP1 mice (P<0.05). Moreover, during the Ang-II infusion, the pressor response to the CGRP receptor antagonist CGRP(8-37) was significantly greater (P<0.05) in hRAMP1 mice (+30+/-2 mm Hg) than in control mice (+19+/-2 mm Hg), confirming a significantly greater antihypertensive action of endogenous CGRP in hRAMP1 mice. We conclude that RAMP1 overexpression attenuates Ang-II-induced hypertension and induces a protective change in cardiovascular autonomic regulation.

The ion channel ASIC2 is required for baroreceptor and autonomic control of the circulation.

Arterial baroreceptors provide a neural sensory input that reflexly regulates the autonomic drive of circulation. Our goal was to test the hypothesis that a member of the acid-sensing ion channel (ASIC) subfamily of the DEG/ENaC superfamily is an important determinant of the arterial baroreceptor reflex. We found that aortic baroreceptor neurons in the nodose ganglia and their terminals express ASIC2. Conscious ASIC2 null mice developed hypertension, had exaggerated sympathetic and depressed parasympathetic control of the circulation, and a decreased gain of the baroreflex, all indicative of an impaired baroreceptor reflex. Multiple measures of baroreceptor activity each suggest that mechanosensitivity is diminished in ASIC2 null mice. The results define ASIC2 as an important determinant of autonomic circulatory control and of baroreceptor sensitivity. The genetic disruption of ASIC2 recapitulates the pathological dysautonomia seen in heart failure and hypertension and defines a molecular defect that may be relevant to its development.