Optimization of mouse kidney digestion protocols for single-cell applications.

Single-cell technologies such as flow cytometry and single-cell RNA sequencing (scRNAseq) have allowed for comprehensive characterisation of the kidney cellulome. However, there is disparity in the various protocols for preparing kidney single-cell suspensions. We aimed to address this limitation by characterising kidney cellular heterogeneity using three previously published single-cell preparation protocols. Single-cell suspensions were prepared from male and female C57BL/6 kidneys using the following kidney tissue dissociation protocols: (P1) a scRNAseq protocol; (P2) a multi-tissue digestion kit from Miltenyi Biotec; and (P3) a protocol established in our laboratory. Following dissociation, flow cytometry was used to identify known major cell types including leukocytes (myeloid and lymphoid), vascular cells (smooth-muscle and endothelial), nephron epithelial cells (intercalating, principal, proximal and distal tubule cells), podocytes, and fibroblasts. Of the protocols tested, P2 yielded significantly less leukocytes and type B-intercalating cells compared to the other techniques. P1 and P3 produced similar yields for most cell types, however, endothelial and myeloid-derived cells were significantly enriched using P1. Significant sex differences were detected in only two cell types: granulocytes (increased in males) and smooth muscle cells (increased in females). Future single-cell studies that aim to enrich for specific kidney cell types, may benefit from this comparative analysis.

Aortic Cellular Heterogeneity in Health and Disease: Novel Insights Into Aortic Diseases From Single-Cell RNA Transcriptomic Data Sets.

Aortic diseases such as atherosclerosis, aortic aneurysms, and aortic stiffening are significant complications that can have significant impact on end-stage cardiovascular disease. With limited pharmacological therapeutic strategies that target the structural changes in the aorta, surgical intervention remains the only option for some patients with these diseases. Although there have been significant contributions to our understanding of the cellular architecture of the diseased aorta, particularly in the context of atherosclerosis, furthering our insight into the cellular drivers of disease is required. The major cell types of the aorta are well defined; however, the advent of single-cell RNA sequencing provides unrivaled insights into the cellular heterogeneity of each aortic cell type and the inferred biological processes associated with each cell in health and disease. This review discusses previous concepts that have now been enhanced with recent advances made by single-cell RNA sequencing with a focus on aortic cellular heterogeneity.

Human amnion epithelial cell therapy reduces hypertension-induced vascular stiffening and cognitive impairment.

Vascular inflammation and fibrosis are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. However, current anti-hypertensive drugs do not treat the underlying tissue damage, such as inflammation-associated fibrosis. Human amnion epithelial cells have several properties amenable for treating vascular pathology. This study tested the effect of amnion epithelial cells on vascular pathology and cognitive impairment during hypertension. Male C57Bl6 mice (8-12 weeks) were administered vehicle (saline; n = 58) or angiotensin II (0.7 mg/kg/d, n = 56) subcutaneously for 14 d. After surgery, a subset of mice were injected with 106 amnion epithelial cells intravenously. Angiotensin II infusion increased systolic blood pressure, aortic pulse wave velocity, accumulation of aortic leukocytes, and aortic mRNA expression of collagen subtypes compared to vehicle-infused mice (n = 9-11, P < 0.05). Administration of amnion epithelial cells attenuated these effects of angiotensin II (P < 0.05). Angiotensin II-induced cognitive impairment was prevented by amnion epithelial cell therapy (n = 7-9, P < 0.05). In the brain, amnion epithelial cells modulated some of the inflammatory genes that angiotensin II promoted differential expression of (n = 6, p-adjusted < 0.05). These findings suggest that amnion epithelial cells could be explored as a potential therapy to inhibit vascular pathology and cognitive impairment during hypertension.

Neuromodulation for the management of chronic pelvic pain syndromes: A systematic review.

BACKGROUND: Chronic pelvic pain is a burdensome condition that involves multiple medical sub-specialties and is often difficult to treat. Sacral stimulation for functional bladder disease has been well established, but little large-scale evidence exists regarding utilization of other neuromodulation techniques to treat chronic pelvic pain. Emerging evidence does suggest that neuromodulation is a promising treatment, and we aim to characterize the use and efficacy of such techniques for treating chronic pelvic pain syndromes. MATERIALS AND METHODS: A systematic review of the literature demonstrating the treatment of chronic pelvic pain syndromes with neuromodulation. Abstracts were reviewed and selected for inclusion, including case series, prospective studies, and randomized controlled trials (RCTs). Case studies and publications in abstract only were not included. The reporting for this systematic review follows Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). The literature search was performed using MEDLINE, Embase, Cochrane Library, PubMed, CINAHL, and Scopus. RESULTS: A total of 50 studies were included in this review, three of which were randomized controlled trials, and the remaining were prospective and retrospective case series. The range of pelvic pain conditions treated included interstitial cystitis, peripheral neuralgia, pudendal neuralgia, gastrointestinal pain, urogenital pain, sacroiliac joint pain, and visceral chronic pelvic pain. We reported on outcomes involving pain, functionality, psychosocial improvement, and medication reduction. CONCLUSIONS: Neuromodulation is a growing treatment for various chronic pain syndromes. Peripheral nerve stimulation was the least studied form of stimulation. Posterior tibial nerve stimulation appears to offer short-term benefit, but long-term results are challenging. Sacral nerve stimulation is established for use in functional bladder syndromes and appears to offer pain improvement in these patients as well. Dorsal root ganglion stimulation and spinal cord stimulation have been used for a variety of conditions with promising results. Further studies of homogeneous patient populations are necessary before strong recommendations can be made at this time, although pooled analysis may also be impactful.

The Photothrombotic Model of Ischemic Stroke.

Stroke is a major cause of morbidity worldwide; yet, there is a lack of treatment options to address post-stroke cognitive and motor impairment, thus there is an urgency for developing neuroprotective and restorative therapies. Much of our fundamental understanding of stroke pathology has been derived from animal models. The photothrombotic model of ischemic stroke is commonly used to study cellular and molecular mechanisms of neurodegeneration, test functional/cognitive outcomes, identify important biomarkers, and assess the effectiveness of novel therapies. It allows for the precise targeting of an infarct to a specific region of the brain, has a low mortality rate, low seizure rate, and is relatively easy to perform. This chapter outlines materials and methods for the photothrombotic model of ischemic stroke in mice, its limitations, and some considerations needed when using this model.

Sex-specific effects of a high fat diet on aortic inflammation and dysfunction.

Obesity and vascular dysfunction are independent and sexually dimorphic risk factors for cardiovascular disease. A high fat diet (HFD) is often used to model obesity in mice, but the sex-specific effects of this diet on aortic inflammation and function are unclear. Therefore, we characterized the aortic immune cell profile and function in 6-week-old male and female C57BL/6 mice fed a normal chow diet (NCD) or HFD for 10 weeks. Metabolic parameters were measured weekly and fortnightly. At end point, aortic immune cell populations and endothelial function were characterized using flow cytometry and wire myography. HFD-male mice had higher bodyweight, blood cholesterol, fasting blood glucose and plasma insulin levels than NCD mice (P < 0.05). HFD did not alter systolic blood pressure (SBP), glycated hemoglobin or blood triglycerides in either sex. HFD-females had delayed increases in bodyweight with a transient increase in fasting blood glucose at week 8 (P < 0.05). Flow cytometry revealed fewer proinflammatory aortic monocytes in females fed a HFD compared to NCD. HFD did not affect aortic leukocyte populations in males. Conversely, HFD impaired endothelium-dependent vasorelaxation, but only in males. Overall, this highlights biological sex as a key factor determining vascular disease severity in HFD-fed mice.

The Concise Guide to PHARMACOLOGY 2023/24: Introduction and Other Protein Targets.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and about 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16176. In addition to this overview, in which are identified 'Other protein targets' which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Multiomics analyses reveal dynamic bioenergetic pathways and functional remodeling of the heart during intermittent fasting.

Intermittent fasting (IF) has been shown to reduce cardiovascular risk factors in both animals and humans, and can protect the heart against ischemic injury in models of myocardial infarction. However, the underlying molecular mechanisms behind these effects remain unclear. To shed light on the molecular and cellular adaptations of the heart to IF, we conducted comprehensive system-wide analyses of the proteome, phosphoproteome, and transcriptome, followed by functional analysis. Using advanced mass spectrometry, we profiled the proteome and phosphoproteome of heart tissues obtained from mice that were maintained on daily 12- or 16 hr fasting, every-other-day fasting, or ad libitum control feeding regimens for 6 months. We also performed RNA sequencing to evaluate whether the observed molecular responses to IF occur at the transcriptional or post-transcriptional levels. Our analyses revealed that IF significantly affected pathways that regulate cyclic GMP signaling, lipid and amino acid metabolism, cell adhesion, cell death, and inflammation. Furthermore, we found that the impact of IF on different metabolic processes varied depending on the length of the fasting regimen. Short IF regimens showed a higher correlation of pathway alteration, while longer IF regimens had an inverse correlation of metabolic processes such as fatty acid oxidation and immune processes. Additionally, functional echocardiographic analyses demonstrated that IF enhances stress-induced cardiac performance. Our systematic multi-omics study provides a molecular framework for understanding how IF impacts the heart's function and its vulnerability to injury and disease.

Sympathetic Nervous System and Atherosclerosis.

Atherosclerosis is characterized by the narrowing of the arterial lumen due to subendothelial lipid accumulation, with hypercholesterolemia being a major risk factor. Despite the recent advances in effective lipid-lowering therapies, atherosclerosis remains the leading cause of mortality globally, highlighting the need for additional therapeutic strategies. Accumulating evidence suggests that the sympathetic nervous system plays an important role in atherosclerosis. In this article, we reviewed the sympathetic innervation in the vasculature, norepinephrine synthesis and metabolism, sympathetic activity measurement, and common signaling pathways of sympathetic activation. The focus of this paper was to review the effectiveness of pharmacological antagonists or agonists of adrenoceptors (α1, α2, ß1, ß2, and ß3) and renal denervation on atherosclerosis. All five types of adrenoceptors are present in arterial blood vessels. α1 blockers inhibit atherosclerosis but increase the risk of heart failure while α2 agonism may protect against atherosclerosis and newer generations of ß blockers and ß3 agonists are promising therapies against atherosclerosis; however, new randomized controlled trials are warranted to investigate the effectiveness of these therapies in atherosclerosis inhibition and cardiovascular risk reduction in the future. The role of renal denervation in atherosclerosis inhibition in humans is yet to be established.

Corrigendum: Phase I trial outcome of amnion cell therapy in patients with ischemic stroke (I-ACT).

[This corrects the article DOI: 10.3389/fnins.2023.1153231.].

Inflammasome Activation Mediates Apoptotic and Pyroptotic Death in Astrocytes Under Ischemic Conditions.

Inflammation is a hallmark mechanism of ischemic stroke-induced brain injury. Recent studies have shown that an intracellular multimeric protein complex known as an inflammasome is a key factor for inducing an inflammatory response, and apoptotic and pyroptotic cell death in ischemic stroke. Inflammasome assembly leads to the activation of pro-inflammatory caspases, and the maturation and secretion of pro-inflammatory cytokines IL-1ß and IL-18. While the role of inflammasomes in ischemic stroke-induced neuronal death, and microglial activation and cell death have been established, little is known about the role of inflammasomes in astrocytes under ischemic conditions. In this study, we investigated the expression and activation of inflammasome components in protoplasmic and fibrous astrocytes under ischemic conditions. We found that both protoplasmic and fibrous astrocytes expressed a differential increase in inflammasome protein components, and that their activation promoted maturation of IL-1ß and IL-18, and secretion of IL-1ß, as well as initiating apoptotic and pyroptotic cell death. Pharmacological inhibition of caspase-1 decreased expression of cleaved caspase-1 and production of mature IL-1ß, and protected against inflammasome-mediated apoptotic and pyroptotic cell death. Overall, this study provides novel insights into the role of inflammasome signaling in astrocytes under ischemic conditions.

Protection against brain injury after ischemic stroke by intravenous human amnion epithelial cells in combination with tissue plasminogen activator.

Background: Thrombolytic agents such as tissue plasminogen activator (tPA) are the only drug class approved to treat ischemic stroke and are usually administered within 4.5 h. However, only ~20% of ischemic stroke patients are eligible to receive the therapy. We previously demonstrated that early intravenous administration of human amnion epithelial cells (hAECs) can limit brain inflammation and infarct growth in experimental stroke. Here, we have tested whether hAECs exert cerebroprotective effects in combination with tPA in mice. Methods: Male C57Bl/6 mice were subjected to middle cerebral artery occlusion for 60 min followed by reperfusion. Immediately following reperfusion, vehicle (saline, n = 31) or tPA (10 mg/kg; n = 73) was administered intravenously. After 30 min of reperfusion, tPA-treated mice were injected intravenously with either hAECs (1×106; n = 32) or vehicle (2% human serum albumin; n = 41). A further 15 sham-operated mice were treated with vehicle (n = 7) or tPA + vehicle (n = 8). Mice were designated to be euthanised at 3, 6 or 24 h post-stroke (n = 21, 31, and 52, respectively), and brains were collected to assess infarct volume, blood-brain barrier (BBB) disruption, intracerebral bleeding and inflammatory cell content. Results: There was no mortality within 6 h of stroke onset, but a high mortality occurred in tPA + saline-treated mice between 6 h and 24 h post-stroke in comparison to mice treated with tPA + hAECs (61% vs. 27%, p = 0.04). No mortality occurred within 24 h of sham surgery in mice treated with tPA + vehicle. We focused on early infarct expansion within 6 h of stroke and found that infarction was ~50% larger in tPA + saline- than in vehicle-treated mice (23 ± 3 mm3 vs. 15 ± 2 mm3, p = 0.02) but not in mice receiving tPA + hAECs (13 ± 2 mm3, p < 0.01 vs. tPA + saline) in which intracerebral hAECs were detected. Similar to the profiles of infarct expansion, BBB disruption and intracerebral bleeding in tPA + saline-treated mice at 6 h was 50-60% greater than in vehicle-treated controls (2.6 ± 0.5 vs. 1.6 ± 0.2, p = 0.05) but not after tPA + hAECs treatment (1.7 ± 0.2, p = 0.10 vs. tPA + saline). No differences in inflammatory cell content were detected between treatment groups. Conclusion: When administered following tPA in acute stroke, hAECs improve safety and attenuate infarct growth in association with less BBB disruption and lower 24 h mortality.

Association of constipation with increased risk of hypertension and cardiovascular events in elderly Australian patients.

The association between constipation and cardiovascular risk is unclear. This population-level matched cohort study compared the association of constipation with hypertension and incident cardiovascular events in 541,172 hospitalized patients aged ≥ 60 years. For each constipation admission, one exact age-matched non-constipated admission was randomly selected from all hospitalizations within 2 weeks to form the comparison cohort. The association of constipation with hypertension and cardiovascular events (myocardial infarction, angina, stroke and transient ischemic attack) were analysed using a series of binary logistic regressions adjusting for age, sex, cardiovascular risk factors, gastrointestinal disorders and sociological factors. Patients with constipation had a higher multivariate-adjusted risk for hypertension (odds ratio [OR], 1.96; 95% confidence interval [CI] 1.94-1.99; P < 0.001). Compared to patients with neither constipation nor hypertension, there was a higher multivariate-adjusted risk for cardiovascular events in patients with constipation alone (OR, 1.58; 95% CI 1.55-1.61; P < 0.001) or hypertension alone (OR, 6.12; 95% CI 5.99-6.26; P < 0.001). In patients with both constipation and hypertension, the risk for all cardiovascular events appeared to be additive (OR, 6.53; 95% CI 6.40-6.66; P < 0.001). In conclusion, among hospital patients aged 60 years or older, constipation is linked to an increased risk of hypertension and cardiovascular events. These findings suggest that interventions to address constipation may reduce cardiovascular risk in elderly patients.

Severe Dystrophic Calcification of a Spinal Cord Stimulator Pulse Generator Pocket: A Case Report.

A spinal cord stimulator is an important long-term treatment modality for refractory chronic pain of multiple etiologies. Hardware-related complications remain known adverse events associated with this intervention. Understanding the risk factors for development of such complications is important for optimizing the efficacy and longevity of spinal cord stimulators. This case report highlights an uncommon case of implantable pulse generator site calcification that was discovered incidentally on spinal cord stimulator explant.

Proteasome inhibition reduces plasma cell and antibody secretion, but not angiotensin II-induced hypertension.

Introduction: Depletion of mature B cells affords protection against experimental hypertension. However, whether B cell-mediated hypertension is dependent on differentiation into antibody-secreting cells (ASCs) remains unclear. Using the proteasome inhibitor, bortezomib, the present study tested the effect of ASC reduction on angiotensin II-induced hypertension. Methods: Male C57BL6/J mice were infused with angiotensin II (0.7 mg/kg/day; s.c.) for 28 days via osmotic minipump to induce hypertension. Normotensive control mice received saline infusion. Bortezomib (750 µg/kg) or vehicle (0.1% DMSO) was administered (i.v.) 3 days prior to minipump implantation, and twice weekly thereafter. Systolic blood pressure was measured weekly using tail-cuff plethysmography. Spleen and bone marrow B1 (CD19+B220-), B2 (B220+CD19+) and ASCs (CD138hiSca-1+Blimp-1+) were enumerated by flow cytometry. Serum immunoglobulins were quantified using a bead-based immunoassay. Results: Bortezomib treatment reduced splenic ASCs by ∼68% and ∼64% compared to vehicle treatment in normotensive (2.00 ± 0.30 vs. 0.64 ± 0.15 × 105 cells; n = 10-11) and hypertensive mice (0.52 ± 0.11 vs. 0.14 ± 0.02 × 105 cells; n = 9-11), respectively. Bone marrow ASCs were also reduced by bortezomib in both normotensive (4.75 ± 1.53 vs. 1.71 ± 0.41 × 103 cells; n = 9-11) and hypertensive mice (4.12 ± 0.82 vs. 0.89 ± 0.18 × 103 cells; n = 9-11). Consistent with ASC reductions, bortezomib reduced serum IgM and IgG2a in all mice. Despite these reductions in ASCs and antibody levels, bortezomib did not affect angiotensin II-induced hypertension over 28 days (vehicle: 182 ± 4 mmHg vs. bortezomib: 177 ± 7 mmHg; n = 9-11). Conclusion: Reductions in ASCs and circulating IgG2a and IgM did not ameliorate experimental hypertension, suggesting other immunoglobulin isotypes or B cell effector functions may promote angiotensin II-induced hypertension.

Chronic cerebral hypoperfusion: a critical feature in unravelling the etiology of vascular cognitive impairment.

Vascular cognitive impairment (VCI) describes a wide spectrum of cognitive deficits related to cerebrovascular diseases. Although the loss of blood flow to cortical regions critically involved in cognitive processes must feature as the main driver of VCI, the underlying mechanisms and interactions with related disease processes remain to be fully elucidated. Recent clinical studies of cerebral blood flow measurements have supported the role of chronic cerebral hypoperfusion (CCH) as a major driver of the vascular pathology and clinical manifestations of VCI. Here we review the pathophysiological mechanisms as well as neuropathological changes of CCH. Potential interventional strategies for VCI are also reviewed. A deeper understanding of how CCH can lead to accumulation of VCI-associated pathology could potentially pave the way for early detection and development of disease-modifying therapies, thus allowing preventive interventions instead of symptomatic treatments.

Phase I trial outcome of amnion cell therapy in patients with ischemic stroke (I-ACT).

Background: We proposed a Phase I dose escalation trial to assess the safety of allogeneic human amniotic epithelial cells (hAECs) in stroke patients with a view to informing the design for a Phase II trial. Methods: The design is based on 3 + 3 dose escalation design with additional components for measuring MR signal of efficacy as well as the effect of hAECs (2-8 × 106/kg, i.v.) on preventing immunosuppression after stroke. Results: Eight patients (six males) were recruited within 24 h of ischemic stroke onset and were infused with hAECs. We were able to increase the dose of hAECs to 8 × 106 cells/kg (2 × 106/kg, n = 3; 4 × 106/kg, n = 3; 8 × 106/kg, n = 2). The mean age is 68.0 ± 10.9 (mean ± SD). The frequencies of hypertension and hyperlipidemia were 87.5%, diabetes was 37.5%, atrial fibrillation was 50%, ischemic heart disease was 37.5% and ever-smoker was 25%. Overall, baseline NIHSS was 7.5 ± 3.1, 7.8 ± 7.2 at 24 h, and 4.9 ± 5.4 at 1 week (n = 8). The modified Rankin scale at 90 days was 2.1 ± 1.2. Supplemental oxygen was given in five patients during hAEC infusion. Using pre-defined criteria, two serious adverse events occurred. One patient developed recurrent stroke and another developed pulmonary embolism whilst in rehabilitation. For the last four patients, infusion of hAECs was split across separate infusions on subsequent days to reduce the risk for fluid overload. Conclusion: Our Phase I trial demonstrates that a maximal dose of 2 × 106/kg hAECs given intravenously each day over 2 days (a total of 4 × 106/kg) is safe and optimal for use in a Phase II trial. Clinical trial registration: ClinicalTrials.gov, identifier ACTRN12618000076279P.

The GHB analogue HOCPCA improves deficits in cognition and sensorimotor function after MCAO via CaMKIIα.

Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) is a major contributor to physiological and pathological glutamate-mediated Ca2+ signals, and its involvement in various critical cellular pathways demands specific pharmacological strategies. We recently presented γ-hydroxybutyrate (GHB) ligands as the first small molecules selectively targeting and stabilizing the CaMKIIα hub domain. Here, we report that the cyclic GHB analogue 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA), improves sensorimotor function after experimental stroke in mice when administered at a clinically relevant time and in combination with alteplase. Further, we observed improved hippocampal neuronal activity and working memory after stroke. On the biochemical level, we observed that hub modulation by HOCPCA results in differential effects on distinct CaMKII pools, ultimately alleviating aberrant CaMKII signalling after cerebral ischemia. As such, HOCPCA normalised cytosolic Thr286 autophosphorylation after ischemia in mice and downregulated ischemia-specific expression of a constitutively active CaMKII kinase proteolytic fragment. Previous studies suggest holoenzyme stabilisation as a potential mechanism, yet a causal link to in vivo findings requires further studies. Similarly, HOCPCA's effects on dampening inflammatory changes require further investigation as an underlying protective mechanism. HOCPCA's selectivity and absence of effects on physiological CaMKII signalling highlight pharmacological modulation of the CaMKIIα hub domain as an attractive neuroprotective strategy.

Pathophysiological Links Between Obesity and Dementia.

Obesity is a major global health concern, with prevalence rates rapidly rising due to increased availability of highly processed foods rich in fats and/or sugars and technological advances promoting more sedentary behaviour. There is increasing evidence to suggest that obesity predisposes individuals to developing cognitive impairment and dementia. However, the relationship between the brain and the peripheral metabolic state is complex, and many of the underlying mechanisms of cognitive impairment in obesity are yet to be fully elucidated. To better understand the links between obesity and dementia, further work is required to determine pathological changes occurring in the brain during obesity. In this mini-review, we discuss the role of two pathological features of obesity (the gut-brain axis and systemic inflammation) and their potential contribution to dementia.