Angiotensin-II receptor type Ia does not contribute to cardiac atrophy following high-thoracic spinal cord injury in mice.

NEW FINDINGS: What is the central question of this study? What is the role of the renin-angiotensin system with angiotensin II acting via its receptor AT1a in spinal cord injury-induced cardiac atrophy? What is the main finding and its importance? Knockout of AT1a did not protect mice that had undergone thoracic level 4 transection from cardiac atrophy. There were no histopathological signs but there was reduced load-dependent left ventricular function (lower stroke volume and cardiac output) with preserved ejection fraction. ABSTRACT: Spinal cord injury (SCI) leads to cardiac atrophy often accompanied by functional deficits. The renin-angiotensin system (RAS) with angiotensin II (AngII) signalling via its receptor AT1a might contribute to cardiac atrophy post-SCI. We performed spinal cord transection at thoracic level T4 (T4-Tx) or sham-operation in female wild-type mice (WT, n = 27) and mice deficient in AT1a (Agtr1a-/- , n = 27). Echocardiography (0, 7, 21 and 28 days post-SCI) and histology and gene expression analyses at 1 and 2 months post-SCI were performed. We found cardiac atrophy post-SCI: reduced heart weight, reduced estimated left ventricular mass in Agtr1a-/- , and reduced cardiomyocyte diameter in WT mice. Although, the latter as well as stroke volume (SV) and cardiac output (CO) were reduced in Agtr1a-/- mice already at baseline, cardiomyocyte diameter was even smaller in injured Agtr1a-/- mice compared to injured WT mice. SV and CO were reduced in WT mice post-SCI. Ejection fraction and fractional shortening were preserved post-SCI in both genotypes. There were no histological signs of fibrosis and pathology in the cardiac sections of either genotype post-SCI. Gene expression of Agtr1a showed a trend for up-regulation at 2 months post-SCI; angiotensinogen was up-regulated at 2 month post-SCI in both genotypes. AngII receptor type 2 (Agtr2) was up- and down-regulated at 1 and 2 months post-SCI in WT mice, respectively, and Ang-(1-7) receptor (Mas) at 1 and 2 months post-SCI. Atrogin-1/MAFbx and MuRF1, atrophy markers, were not significantly up-regulated post-SCI. Our data show that lack of AT1a does not protect from cardiac atrophy post-SCI.

Angiotensin-(1-7) Receptor Mas Deficiency Does Not Exacerbate Cardiac Atrophy Following High-Level Spinal Cord Injury in Mice.

Experimental spinal cord injury (SCI) causes a morphological and functional deterioration of the heart, in which the renin-angiotensin system (RAS) might play a role. The recently discovered non-canonical axis of RAS with angiotensin-(1-7) and its receptor Mas, which is associated with cardioprotection could be essential to prevent damage to the heart following SCI. We investigated the cardiac consequences of SCI and the role of Mas in female wild-type (WT, n = 22) and mice deficient of Mas (Mas-/- , n = 25) which underwent spinal cord transection at thoracic level T4 (T4-Tx) or sham-operation by echocardiography (0, 7, 21, and 28 days post-SCI), histology and gene expression analysis at 1 or 2 months post-SCI. We found left ventricular mass reduction with preserved ejection fraction (EF) and fractional shortening in WT as well as Mas-/- mice. Cardiac output was reduced in Mas-/- mice, whereas stroke volume (SV) was reduced in WT T4-Tx mice. Echocardiographic indices did not differ between the genotypes. Smaller heart weight (HW) and smaller cardiomyocyte diameter at 1 month post-SCI compared to sham mice was independent of genotype. The muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1 were upregulated or showed a trend for upregulation in WT mice at 2 months post-SCI, respectively. Angiotensinogen gene expression was upregulated at 1 month post-SCI and angiotensin II receptor type 2 downregulated at 2 month post-SCI in Mas-/- mice. Mas was downregulated post-SCI. Cardiac atrophy following SCI, not exacerbated by lack of Mas, is a physiological reaction as there were no signs of cardiac pathology and dysfunction.

Distinct roles of angiotensin receptors in autonomic dysreflexia following high-level spinal cord injury in mice.

Autonomic dysreflexia (AD), a syndrome caused by loss of supraspinal control over sympathetic activity and amplified vascular reflex upon sensory stimuli below injury level, is a major health problem in high-level spinal cord injury (SCI). After supraspinal sympathetic control of the vasculature below the lesion is lost, the renin-angiotensin system (RAS) is thought to be involved in AD by regulating blood pressure and vascular reactivity. In this study, we aimed to assess the role of different RAS receptors during AD following SCI. Therefore, we induced AD by colorectal distention (CRD) in wild-type mice and mice deficient in the RAS components angiotensin (Ang) II type 1a receptor (AT1a) (Agtr1a-/-) and Ang-(1-7) receptor Mas (Mas-/-) four weeks after complete transection of spinal cord at thoracic level 4 (T4). Systemic blood pressure measurements and wire myography technique were performed to assess hemodynamics and the reactivity of peripheral arteries, respectively. CRD increased mean arterial blood pressure (MAP) and decreased heart rate (HR) in all three animal groups. However, we found less increases in MAP in Mas-/- mice compared to control mice after CRD, whereas AT1a deficiency did not affect the hemodynamic response. We found that the reactivity of wild-type and Mas-/- mesenteric arteries, which are innervated from ganglia distal but close to thoracic level T4, was diminished in response to Ang II in AD after T4-SCI, but this difference was not observed in the absence of AT1a receptors. CRD did not influence the reactivity of femoral arteries which are innervated from ganglia more distal to thoracic level T4, in response to Ang II in AD. In conclusion, we identified a specific role of the Ang-(1-7) receptor Mas in regulating the systemic blood pressure increase in AD in T4-SCI mice. Furthermore, AT1a signaling is not involved in this hemodynamic response, but underlies increased vascular reactivity in mesenteric arteries in response to Ang II, where it may contribute to adaptive changes in regional blood flow.

Angiotensin-(1-7) Receptor Mas in Hemodynamic and Thermoregulatory Dysfunction After High-Level Spinal Cord Injury in Mice: A Pilot Study.

Spinal cord injury (SCI) above mid-thoracic levels leads to autonomic dysfunction affecting both the cardiovascular system and thermoregulation. The renin-angiotensin system (RAS) which is a potent regulator of blood pressure, including its novel beneficial arm with the receptor Mas could be an interesting target in post-SCI hemodynamics. To test the hypothesis that hemodynamics, activity and diurnal patterns of those are more affected in the Mas deficient mice post-SCI we used a mouse model of SCI with complete transection of spinal cord at thoracic level 4 (T4-Tx) and performed telemetric monitoring of blood pressure (BP) and heart rate (HR). Our data revealed that hypothermia deteriorated physiological BP and HR control. Preserving normothermia by keeping mice at 30°C prevented severe hypotension and bradycardia post-SCI. Moreover, it facilitated rapid return of diurnal regulation of BP, HR and activity in wild type (WT) mice. In contrast, although Mas deficient mice had comparable reacquisition of diurnal HR rhythm, they showed delayed recovery of diurnal rhythmicity in BP and significantly lower nocturnal activity. Exposing mice with T4-Tx (kept in temperature-controlled cages) to 23°C room temperature for one hour at different time-points post-SCI, demonstrated their inability to maintain core body temperature, Mas deficient mice being significantly more impaired than WT littermates. We conclude that Mas deficient mice were more resistant to acute hypotension, delayed nocturnal recovery, lower activity and more severely impaired thermoregulation. The ambient temperature had significant effect on hemodynamics and, thus it should be taken into account when assessing cardiovascular parameters post-SCI in mice.

Adverse left ventricular remodeling by glycoprotein nonmetastatic melanoma protein B in myocardial infarction.

Cardiac diseases are the leading cause of death. Available treatment approaches are not sufficient to reverse persistent cardiac damage after injury; thus, the search for new therapeutic targets is essential. Our microarray-based screening in rat hearts 24 h after myocardial infarction (MI) yielded glycoprotein nonmetastatic melanoma protein B (GPNMB), which is known to be involved in inflammation and fibrosis after tissue injury. However, its role in the heart was elusive. We found increased cardiac expression levels of GPNMB in rats and mice after MI. Analysis of DBA/2J mice, which lack functional GPNMB due to a spontaneous point mutation, showed that systemic GPNMB deficiency was associated with preserved cardiac function and less left ventricular dilation after MI compared with DBA/2J mice with reconstituted GPNMB expression. These improvements were associated with decreased expression of matrix metalloproteinase 9, the cardiac stress genes for natriuretic peptides (atrial natriuretic peptide and brain natriuretic peptide), and ß-myosin heavy chain after MI. Moreover, GPNMB deficiency attenuated the dilated cardiomyopathy in muscle lim protein knockout mice but could not prevent cardiac hypertrophy induced by isoprenaline infusion. This is the first experimental study to show that GPNMB adversely influences myocardial remodeling.-Järve, A., Mühlstedt, S., Qadri, F., Nickl, B., Schulz, H., Hübner, N., Özcelik, C., Bader, M. Adverse left ventricular remodeling by glycoprotein nonmetastatic melanoma protein B in myocardial infarction.

Cardiomyocyte-derived CXCL12 is not involved in cardiogenesis but plays a crucial role in myocardial infarction.

UNLABELLED: The chemokine CXCL12/SDF-1 is crucial for heart development and affects cardiac repair processes due to its ability to attract leukocytes and stem cells to injured myocardium. However, there is a great controversy whether CXCL12 is beneficial or detrimental after myocardial infarction (MI). The divergence in the reported CXCL12 actions may be due to the cellular source and time of release of the chemokine after MI. This study was designed to evaluate the role of cardiomyocyte-derived CXCL12 for cardiogenesis and heart repair after MI. We generated two rodent models each targeting CXCL12 in only one cardiac cell type: cardiomyocyte-specific CXCL12-overexpressing transgenic (Tg) rats and CXCL12 conditional knockout (cKO) mice. Animals of both models did not show any signs of cardiac abnormalities under baseline conditions. After induction of MI, cKO mice displayed preserved cardiac function and remodeling. Moreover, fibrosis was less pronounced in the hearts of cKO mice after MI. Accordingly, CXCL12 Tg rats revealed impaired cardiac function post-MI accompanied by enhanced fibrosis. Furthermore, we observed decreased numbers of infiltrating Th1 cells in the hearts of cKO mice. Collectively, our findings demonstrate that cardiomyocyte-derived CXCL12 is not involved in cardiac development but has adverse effects on the heart after injury via promotion of inflammation and fibrosis. KEY MESSAGES: • CXCL12 in cardiomyocytes is not involved in cardiac development. • CXCL12 deficiency in cardiomyocytes improves outcome of myocardial infarction. • CXCL12 overexpression in cardiomyocytes worsens outcome of myocardial infarction. • CXCL12 increases fibrosis and invasion of Th1 cells in the heart after infarction.

FRET imaging of cells transfected with siRNA/liposome complexes.

By monitoring the efficiency of fluorescence resonance energy transfer of dyes attached to the different strands of siRNA, the structural integrity of the latter can be traced inside cells. Here, the experimental details of dye-labeled siRNA construction, tissue culture, and transfection with liposomally formulated siRNAs are given, as well as the conditions for confocal microscopy and an algorithm allowing the visualization of intact siRNA after image data treatment. The method allows rapid screening of different liposomal siRNA formulations, obtained by small scale dual asymmetric centrifugation with high entrapping efficiency.

SDF-1 stimulates neurite growth on inhibitory CNS myelin.

Impaired axonal regeneration is a common observation after central nervous system (CNS) injury. The stromal cell-derived factor-1, SDF-1/CXCL12, has previously been shown to promote axonal growth in the presence of potent chemorepellent molecules known to be important in nervous system development. Here, we report that treatment with SDF-1alpha is sufficient to overcome neurite outgrowth inhibition mediated by CNS myelin towards cultured postnatal dorsal root ganglion neurons. While we found both cognate SDF-1 receptors, CXCR4 and CXCR7/RDC1, to be coexpressed on myelin-sensitive dorsal root ganglion neurons, the distinct expression pattern of CXCR4 on growth cones and branching points of neurites suggests a function of this receptor in chemokine-mediated growth promotion and/or arborization. These in vitro findings were further corroborated as local intrathecal infusion of SDF-1 into spinal cord injury following thoracic dorsal hemisection resulted in enhanced sprouting of corticospinal tract axons into white and grey matter. Our findings indicate that SDF-1 receptor activation might constitute a novel therapeutic approach to promote axonal growth in the injured CNS.

Surveillance of siRNA integrity by FRET imaging.

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.

Glycogen synthase kinase 3beta (GSK3beta) regulates differentiation and proliferation in neural stem cells from the rat subventricular zone.

On the basis of its inhibition by SB216763, we identified the multifunctional enzyme Glycogen Synthase Kinase 3beta (GSK3beta) as a central regulator for differentiation and cell survival of adult neural stem cells. Detected by proteomic approaches, members of the Wnt/beta-catenin signaling pathway appear to participate in enhanced neuronal differentiation and activated transcription of beta-catenin target genes during GSK3beta inhibition, associated with decreased apoptosis.

Human vascular endothelial growth factor protects axotomized retinal ganglion cells in vivo by activating ERK-1/2 and Akt pathways.

Based on its trophic effects on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for the treatment of neurodegenerative diseases. To evaluate the therapeutic potential of VEGF, we here examined effects of this growth factor on the degeneration of axotomized retinal ganglion cells (RGCs), which, as CNS-derived neurons, offer themselves in an excellent way to study neuroprotection in vivo. Making use of a transgenic mouse line that constitutively expresses human VEGF under a neuron-specific enolase promoter, we show that (1) the VEGF-transgenic retina overexpresses human VEGF, (2) RGCs carry the VEGF receptor-2, and (3) vascular networks in normal and axotomized VEGF-transgenic (tg) retinas do not differ from control animals. After axotomy, RGCs of VEGF-tg mice were protected against delayed degeneration, as compared with wild-type littermates. Western blots revealed increased phosphorylated ERK-1/2 and Akt and reduced phosphorylated p38 and activated caspase-3 levels in axotomized VEGF-transgenic retinas. Intravitreous injections of pharmacological ERK-1/2 (PD98059) or Akt (LY294002) inhibitors showed that VEGF exerts neuroprotection by dual activation of ERK-1/2 and Akt pathways. In view that axotomy-induced RGC death occurs slowly and considering that RGCs are CNS-derived neurons, we predict the clinical implementation of VEGF in neurodegenerative diseases of both brain and retina.