BACKGROUND AND PURPOSE Inflammasomes are multimeric complexes that facilitate caspase-1-mediated ... more BACKGROUND AND PURPOSE Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1β and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH Wild-type and inflammasome-deficient ASC −/− mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry.
Introduction: Evidence suggests that hypertension involves underlying inflammation, however wheth... more Introduction: Evidence suggests that hypertension involves underlying inflammation, however whether atherosclerosis - a chronic inflammatory condition - can cause hypertension is unknown. We tested whether blood pressure (BP) is higher in high-fat fed ApoE-/- vs chow-fed wild-type (WT) mice, and whether advanced atherosclerosis is associated with systemic and end-organ inflammation, oxidative stress and endothelial dysfunction. Methods: Male ApoE-/- and WT mice were placed on high fat and chow diets, respectively, from 5-56 weeks. To clarify the effects of ageing alone, aged WT mice were compared to young chow-fed WT mice (8-12 week old). We measured systolic BP, plasma cytokine levels, mRNA expression of inflammatory markers, vascular superoxide and endothelial function. Results: There was no difference in BP of aged ApoE-/- (104.2 ± 2.9 mmHg) and age-matched WT mice (113.2 ± 1.3 mmHg) (n=13-18, P>0.05). However, plasma IL-6, TNF-α and IFN-γ were elevated in ApoE-/- by more than 2-fold vs age-matched WT (n=9-10, all P<0.05), as was brain expression of IL-1β, IL-6, TNF-α, IFN-γ, TGFβ1, CCR2, CCL2, CCL7, CCL8, CCL12 and IL-10 (n=9-10, all P<0.05), and aortic expression of IL-6, CCR2, CCL8 and CCL12 (n=6-8, all P<0.05). Ageing, but not atherosclerosis, increased renal expression of IL-1β, IL-6, TNF-α, CCR2, CCL2, CCL7, CCL8, CCL12 and Foxp3, and aortic expression of CCL2, IL-10 and Foxp3 by at least 2-fold (n=6-10, all P<0.05). In ApoE-/- aorta, Nox2-dependent superoxide production was 4-fold greater than in WT (n=5-6, P<0.05), and endothelium-dependent vasorelaxation to carbachol was markedly reduced by more than half (n=5-7, P<0.05). Ageing alone had no effect on BP, systemic inflammation or endothelial function. Conclusions: Despite the systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not result in elevated BP.
Although hypertension may involve underlying inflammation, it is unknown whether advanced atheros... more Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosisa chronic inflammatory conditioncan by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end-organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE-/and wild-type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL-6, TNF-α, IFN-γ and/or IL-1β) was elevated in plasma, brain, and aorta of ApoE-/mice. Aortic superoxide production was ~3.5-fold greater, and endothelium-dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE-/versus wild-type mice. There was no difference in blood pressure of aged ApoE-/-(104±3 mmHg, n=13) and wild-type mice (113±1 mmHg, n=18). To clarify any effects of aging alone, findings from 61 week-old wild-type mice were compared with those from young (8-12 weeks old) chow-fed wild-type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL-1β). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.
Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological... more Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological and immunological antecedents, however the relationship between these is unclear. Methods and Results: Here we used a mouse model of diet-induced obesity, pressure myography, immunohistochemistry, flow cytometry and radiotelemtry to demonstrate that immune cells, located within the vascular adventitia of arteries important for controlling blood pressure, produce nerve growth factor (NGF) and rapidly initiate growth of the perivascular sympathetic nerve plexus; which increases vascular tone and thereby elevates blood pressure. We also show that Rag1-/- mice lacking T and B cells do not develop hypertension when obesity is induced with a high fat diet (HFD) and show that their sympathetic innervation and vascular tone remains normal, suggesting that neurogenic hypertension in the obese likely occurs independently of weight gain. Furthermore, we show that the adoptive transfer of wildtype CD3+ T cells restores the sympathetic hyperinnervation and associated hypertension whereas the transfer of CD3+ T cells deficient in NGF had no effect on sympathetic innervation or blood pressure. Conclusion: These results not only demonstrate the role of T cells in the genesis of obesity-related hypertension but also demonstrate the pivotal role of NGF in linking dysfunction of the immune and sympathetic nervous systems that characterize hypertension in the obese. a, Change in body weight b, nerve-mediated contraction amplitude recorded from pressurized small mesenteric arteries (1Hz, 30s) and c, systolic BP in mice in RAG-1-/- mice, which lack B and T cells, fed NCD or HFD. RAG-1-/-Tcell mice are recipients of CD3+ T cells via adoptive transfer. d, Perivascular nerve density (synaptophysin) over wholemount small mesenteric arteries from RAG-1-/- mice. Scale, 50μm.
Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapie... more Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapies. To overcome this, we demonstrated that the anti-fibrotic drug, serelaxin (recombinant human relaxin; RLX), could prevent renal fibrosis progression in a unilateral ureteric obstruction (UUO)-induced murine model of interstitial renal disease and improve the ability of injected human bone marrow-derived mesenchymal stem cells (BM-MSCs) to attenuate renal injury. To advance the therapeutic potential of this combination therapy, we have now investigated its effects as an intervention therapy in the UUO model, and in a uninephrectomised/deoxycorticosterone acetate/drinking saline water (1K/DOCA/salt) model of hypertension. 7-8 week old male C57B6J mice were subjected to UUO or 1K/DOCA/salt (2.4mg/day, s.c; 21 days). Sub-groups of injured mice (n=6/group) were administered RLX (0.5mg/kg/day; s.c) alone, BM-MSCs (1x10 6 /mouse; i.v) alone or both combined 3-days post-UUO or 14 days post-1K/DOCA/salt-treatment. Untreated injured mice and sham-operated or 1K-mice were included as respective controls. Mice were killed 10 days post-UUO or 21 days post-1K/DOCA/salt treatment (7 days post-intervention) for tissue collection and analysis. UUO- and 1K/DOCA/salt-injured mice underwent significant renal damage, inflammation and (interstitial and total collagen-induced) fibrosis at the time-points studied, as determined by histological and biochemical analyses (all p<0.01 vs respective uninjured controls). All 1K/DOCA/salt-treated mice had systolic blood pressure levels of 140-150mmHg over the experimental period, compared to 1K-mice having 115-120mmHg (as measured by tail cuff plethysmography; p<0.01). Unlike BM-MSCs alone, the combined effects of BM-MSCs and RLX significantly reduced established UUO-induced renal damage and fibrosis, primarily by reducing myofibroblast-induced collagen deposition (all p<0.05 vs BM-MSCs alone). Treatment-induced effects of the hypertensive model are currently being evaluated and will be presented. These findings confirm that agents that can effectively reduce established fibrosis can enhance the therapeutic potential of BM-MSCs in treating kidney diseases, when added in combination.
Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow ... more Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 μg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.
Renal inflammation, leading to fibrosis and impaired function is a major contributor to the devel... more Renal inflammation, leading to fibrosis and impaired function is a major contributor to the development of hypertension. The NLRP3 inflammasome mediates inflammation in several chronic diseases by processing the cytokines pro-interleukin (IL)-1b and pro-IL-18. In this study, we investigated whether MCC950, a recently-identified inhibitor of NLRP3 activity, reduces blood pressure (BP), renal inflammation, fibrosis and dysfunction in mice with established hypertension.
Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have emerged as a promising treatme... more Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have emerged as a promising treatment option for chronic kidney disease (CKD), based on their anti-hypertensive, immunomodulatory and tissue-reparative effects. However, the build-up of fibrosis (scar tissue) in CKD significantly impairs the viability and efficacy of implanted BM-MSCs. To address this, we previously combined the separate administration of BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), which enhanced the renoprotective effects of BM-MSCs in normotensive and hypertensive models of CKD. To advance the clinical application of this combined therapy, we have now engineered BM-MSCs to express RLX and GFP (BM-MSCs-RLX+GFP) or GFP alone (BM-MSCs-GFP) using lentiviral transduction, to deliver the combined treatments via the same route simultaneously. The therapeutic effects of BM-MSCs-RLX+GFP were then investigated in a high salt (HS; 2% NaCI)-induced model of hypertension. Flow cytometry revealed a transduc...
Introduction: Evidence suggests that hypertension involves underlying inflammation, however wheth... more Introduction: Evidence suggests that hypertension involves underlying inflammation, however whether atherosclerosis - a chronic inflammatory condition - can cause hypertension is unknown. We tested whether blood pressure (BP) is higher in high-fat fed ApoE-/- vs chow-fed wild-type (WT) mice, and whether advanced atherosclerosis is associated with systemic and end-organ inflammation, oxidative stress and endothelial dysfunction. Methods: Male ApoE-/- and WT mice were placed on high fat and chow diets, respectively, from 5-56 weeks. To clarify the effects of ageing alone, aged WT mice were compared to young chow-fed WT mice (8-12 week old). We measured systolic BP, plasma cytokine levels, mRNA expression of inflammatory markers, vascular superoxide and endothelial function. Results: There was no difference in BP of aged ApoE-/- (104.2 ± 2.9 mmHg) and age-matched WT mice (113.2 ± 1.3 mmHg) (n=13-18, P>0.05). However, plasma IL-6, TNF-α and IFN-γ were elevated in ApoE-/- by more than...
Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological... more Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological and immunological antecedents, however the relationship between these is unclear. Methods and Results: Here we used a mouse model of diet-induced obesity, pressure myography, immunohistochemistry, flow cytometry and radiotelemtry to demonstrate that immune cells, located within the vascular adventitia of arteries important for controlling blood pressure, produce nerve growth factor (NGF) and rapidly initiate growth of the perivascular sympathetic nerve plexus; which increases vascular tone and thereby elevates blood pressure. We also show that Rag1-/- mice lacking T and B cells do not develop hypertension when obesity is induced with a high fat diet (HFD) and show that their sympathetic innervation and vascular tone remains normal, suggesting that neurogenic hypertension in the obese likely occurs independently of weight gain. Furthermore, we show that the adoptive transfer of wildtype ...
Inflammasomes are a family of interleukin-1 processing complexes and master regulators of inflamm... more Inflammasomes are a family of interleukin-1 processing complexes and master regulators of inflammation. We recently showed that both one-kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)- and angiotensin II-dependent hypertension in mice are associated with elevated expression of the NLRP3 inflammasome in the kidneys. Moreover, genetic deficiency of a key subunit critical for NLRP3 inflammasome activity protected mice against renal inflammation and chronic pressor responses associated with these models. As a step towards translation of these findings into new therapies, here we investigated whether a highly specific small-molecule inhibitor of NLRP3 inflammasome activity, MCC950, similarly reduces the deleterious effects of 1K/DOCA/salt on blood pressure (BP) and renal function. Male C57BL6/J mice were implanted with telemetry probes for continuous recording of BP (mean arterial (MAP), systolic, diastolic) and heart rate (HR), or placed in metabolic cages for 24 h urine collect...
Background and PurposeOxidative stress and fibrosis are hallmarks of cardiomyopathy‐induced heart... more Background and PurposeOxidative stress and fibrosis are hallmarks of cardiomyopathy‐induced heart failure yet are not effectively targeted by current frontline therapies. Here, the therapeutic effects of the anti‐oxidant, N‐acetylcysteine (NAC), were compared and combined with an acute heart failure drug with established anti‐fibrotic effects, serelaxin (RLX), in a murine model of cardiomyopathy.Experimental ApproachAdult male 129sv mice were subjected to repeated isoprenaline (25 mg·kg−1)‐induced cardiac injury for five consecutive days and then left to undergo fibrotic healing until Day 14. Subgroups of isoprenaline‐injured mice were treated with RLX (0.5 mg·kg−1·day−1), NAC (25 mg·kg−1·day−1) or both combined, given subcutaneously via osmotic minipumps from Day 7 to 14. Control mice received saline instead of isoprenaline.Key ResultsIsoprenaline‐injured mice showed increased left ventricular (LV) inflammation (~5‐fold), oxidative stress (~1–2.5‐fold), cardiomyocyte hypertrophy (~...
Interleukin-18 (IL-18) is a pro-inflammatory cytokine released downstream of inflammasome activat... more Interleukin-18 (IL-18) is a pro-inflammatory cytokine released downstream of inflammasome activation that is elevated in hypertensive patients. However, whether IL-18 plays a causal role in hyperte...
IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood p... more IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood pressure and end-organ damage is unknown. We examined the role of IL-18 in the development of renal inflammation and injury in a mouse model of low-renin hypertension. Hypertension was induced in male C57BL6/J (WT) and IL-18−/−mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% drinking saline (1K/DOCA/salt). Normotensive controls received uninephrectomy and placebo (1K/placebo). Blood pressure was measured via tail cuff or radiotelemetry. After 21 days, kidneys were harvested for (immuno)histochemical, quantitative-PCR and flow cytometric analyses of fibrosis, inflammation, and immune cell infiltration. 1K/DOCA/salt-treated WT mice developed hypertension, renal fibrosis, upregulation of proinflammatory genes, and accumulation of CD3+T cells in the kidneys. They also displayed increased expression of IL-18 on tubular epithelial cells. IL-18−/−mice were profoundly...
Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow ... more Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 μg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.
Background and PurposeFibrosis is a hallmark of chronic kidney disease (CKD) that significantly c... more Background and PurposeFibrosis is a hallmark of chronic kidney disease (CKD) that significantly contributes to renal dysfunction, and impairs the efficacy of stem cell‐based therapies. This study determined whether combining bone marrow‐derived mesenchymal stem cells (BM‐MSCs) with the renoprotective effects of recombinant human relaxin (serelaxin) could therapeutically reduce renal fibrosis in mice with one kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)‐induced hypertension, compared with the effects of the ACE inhibitor, perindopril.Experimental ApproachAdult male C57BL/6 mice were uni‐nephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt) for 21 days. Control mice were uni‐nephrectomised but received water over the same time period. Sub‐groups of 1K/DOCA/salt‐injured mice (n = 5–8 per group) were treated with either serelaxin (0.5 mg·kg−1·day−1) or BM‐MSCs (1 × 106 per mouse) alone; both treatments combined (with 0.5 × 106 or 1 × 106 BM...
Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapie... more Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapies. To overcome this, we demonstrated that the anti-fibrotic drug, serelaxin (recombinant human relaxin; RLX), could prevent renal fibrosis progression in a unilateral ureteric obstruction (UUO)-induced murine model of interstitial renal disease and improve the ability of injected human bone marrow-derived mesenchymal stem cells (BM-MSCs) to attenuate renal injury. To advance the therapeutic potential of this combination therapy, we have now investigated its effects as an intervention therapy in the UUO model, and in a uninephrectomised/deoxycorticosterone acetate/drinking saline water (1K/DOCA/salt) model of hypertension. 7-8 week old male C57B6J mice were subjected to UUO or 1K/DOCA/salt (2.4mg/day, s.c; 21 days). Sub-groups of injured mice (n=6/group) were administered RLX (0.5mg/kg/day; s.c) alone, BM-MSCs (1x10 6 /mouse; i.v) alone or both combined 3-days post-UUO or 14 days post-1K/...
To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal infl... more To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05)...
BACKGROUND AND PURPOSE Inflammasomes are multimeric complexes that facilitate caspase-1-mediated ... more BACKGROUND AND PURPOSE Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1β and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. EXPERIMENTAL APPROACH Wild-type and inflammasome-deficient ASC −/− mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry.
Introduction: Evidence suggests that hypertension involves underlying inflammation, however wheth... more Introduction: Evidence suggests that hypertension involves underlying inflammation, however whether atherosclerosis - a chronic inflammatory condition - can cause hypertension is unknown. We tested whether blood pressure (BP) is higher in high-fat fed ApoE-/- vs chow-fed wild-type (WT) mice, and whether advanced atherosclerosis is associated with systemic and end-organ inflammation, oxidative stress and endothelial dysfunction. Methods: Male ApoE-/- and WT mice were placed on high fat and chow diets, respectively, from 5-56 weeks. To clarify the effects of ageing alone, aged WT mice were compared to young chow-fed WT mice (8-12 week old). We measured systolic BP, plasma cytokine levels, mRNA expression of inflammatory markers, vascular superoxide and endothelial function. Results: There was no difference in BP of aged ApoE-/- (104.2 ± 2.9 mmHg) and age-matched WT mice (113.2 ± 1.3 mmHg) (n=13-18, P&amp;gt;0.05). However, plasma IL-6, TNF-α and IFN-γ were elevated in ApoE-/- by more than 2-fold vs age-matched WT (n=9-10, all P&amp;lt;0.05), as was brain expression of IL-1β, IL-6, TNF-α, IFN-γ, TGFβ1, CCR2, CCL2, CCL7, CCL8, CCL12 and IL-10 (n=9-10, all P&amp;lt;0.05), and aortic expression of IL-6, CCR2, CCL8 and CCL12 (n=6-8, all P&amp;lt;0.05). Ageing, but not atherosclerosis, increased renal expression of IL-1β, IL-6, TNF-α, CCR2, CCL2, CCL7, CCL8, CCL12 and Foxp3, and aortic expression of CCL2, IL-10 and Foxp3 by at least 2-fold (n=6-10, all P&amp;lt;0.05). In ApoE-/- aorta, Nox2-dependent superoxide production was 4-fold greater than in WT (n=5-6, P&amp;lt;0.05), and endothelium-dependent vasorelaxation to carbachol was markedly reduced by more than half (n=5-7, P&amp;lt;0.05). Ageing alone had no effect on BP, systemic inflammation or endothelial function. Conclusions: Despite the systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not result in elevated BP.
Although hypertension may involve underlying inflammation, it is unknown whether advanced atheros... more Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosisa chronic inflammatory conditioncan by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end-organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE-/and wild-type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL-6, TNF-α, IFN-γ and/or IL-1β) was elevated in plasma, brain, and aorta of ApoE-/mice. Aortic superoxide production was ~3.5-fold greater, and endothelium-dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE-/versus wild-type mice. There was no difference in blood pressure of aged ApoE-/-(104±3 mmHg, n=13) and wild-type mice (113±1 mmHg, n=18). To clarify any effects of aging alone, findings from 61 week-old wild-type mice were compared with those from young (8-12 weeks old) chow-fed wild-type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL-1β). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.
Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological... more Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological and immunological antecedents, however the relationship between these is unclear. Methods and Results: Here we used a mouse model of diet-induced obesity, pressure myography, immunohistochemistry, flow cytometry and radiotelemtry to demonstrate that immune cells, located within the vascular adventitia of arteries important for controlling blood pressure, produce nerve growth factor (NGF) and rapidly initiate growth of the perivascular sympathetic nerve plexus; which increases vascular tone and thereby elevates blood pressure. We also show that Rag1-/- mice lacking T and B cells do not develop hypertension when obesity is induced with a high fat diet (HFD) and show that their sympathetic innervation and vascular tone remains normal, suggesting that neurogenic hypertension in the obese likely occurs independently of weight gain. Furthermore, we show that the adoptive transfer of wildtype CD3+ T cells restores the sympathetic hyperinnervation and associated hypertension whereas the transfer of CD3+ T cells deficient in NGF had no effect on sympathetic innervation or blood pressure. Conclusion: These results not only demonstrate the role of T cells in the genesis of obesity-related hypertension but also demonstrate the pivotal role of NGF in linking dysfunction of the immune and sympathetic nervous systems that characterize hypertension in the obese. a, Change in body weight b, nerve-mediated contraction amplitude recorded from pressurized small mesenteric arteries (1Hz, 30s) and c, systolic BP in mice in RAG-1-/- mice, which lack B and T cells, fed NCD or HFD. RAG-1-/-Tcell mice are recipients of CD3+ T cells via adoptive transfer. d, Perivascular nerve density (synaptophysin) over wholemount small mesenteric arteries from RAG-1-/- mice. Scale, 50μm.
Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapie... more Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapies. To overcome this, we demonstrated that the anti-fibrotic drug, serelaxin (recombinant human relaxin; RLX), could prevent renal fibrosis progression in a unilateral ureteric obstruction (UUO)-induced murine model of interstitial renal disease and improve the ability of injected human bone marrow-derived mesenchymal stem cells (BM-MSCs) to attenuate renal injury. To advance the therapeutic potential of this combination therapy, we have now investigated its effects as an intervention therapy in the UUO model, and in a uninephrectomised/deoxycorticosterone acetate/drinking saline water (1K/DOCA/salt) model of hypertension. 7-8 week old male C57B6J mice were subjected to UUO or 1K/DOCA/salt (2.4mg/day, s.c; 21 days). Sub-groups of injured mice (n=6/group) were administered RLX (0.5mg/kg/day; s.c) alone, BM-MSCs (1x10 6 /mouse; i.v) alone or both combined 3-days post-UUO or 14 days post-1K/DOCA/salt-treatment. Untreated injured mice and sham-operated or 1K-mice were included as respective controls. Mice were killed 10 days post-UUO or 21 days post-1K/DOCA/salt treatment (7 days post-intervention) for tissue collection and analysis. UUO- and 1K/DOCA/salt-injured mice underwent significant renal damage, inflammation and (interstitial and total collagen-induced) fibrosis at the time-points studied, as determined by histological and biochemical analyses (all p&amp;amp;lt;0.01 vs respective uninjured controls). All 1K/DOCA/salt-treated mice had systolic blood pressure levels of 140-150mmHg over the experimental period, compared to 1K-mice having 115-120mmHg (as measured by tail cuff plethysmography; p&amp;amp;lt;0.01). Unlike BM-MSCs alone, the combined effects of BM-MSCs and RLX significantly reduced established UUO-induced renal damage and fibrosis, primarily by reducing myofibroblast-induced collagen deposition (all p&amp;amp;lt;0.05 vs BM-MSCs alone). Treatment-induced effects of the hypertensive model are currently being evaluated and will be presented. These findings confirm that agents that can effectively reduce established fibrosis can enhance the therapeutic potential of BM-MSCs in treating kidney diseases, when added in combination.
Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow ... more Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 μg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.
Renal inflammation, leading to fibrosis and impaired function is a major contributor to the devel... more Renal inflammation, leading to fibrosis and impaired function is a major contributor to the development of hypertension. The NLRP3 inflammasome mediates inflammation in several chronic diseases by processing the cytokines pro-interleukin (IL)-1b and pro-IL-18. In this study, we investigated whether MCC950, a recently-identified inhibitor of NLRP3 activity, reduces blood pressure (BP), renal inflammation, fibrosis and dysfunction in mice with established hypertension.
Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have emerged as a promising treatme... more Human bone marrow-derived mesenchymal stromal cells (BM-MSCs) have emerged as a promising treatment option for chronic kidney disease (CKD), based on their anti-hypertensive, immunomodulatory and tissue-reparative effects. However, the build-up of fibrosis (scar tissue) in CKD significantly impairs the viability and efficacy of implanted BM-MSCs. To address this, we previously combined the separate administration of BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), which enhanced the renoprotective effects of BM-MSCs in normotensive and hypertensive models of CKD. To advance the clinical application of this combined therapy, we have now engineered BM-MSCs to express RLX and GFP (BM-MSCs-RLX+GFP) or GFP alone (BM-MSCs-GFP) using lentiviral transduction, to deliver the combined treatments via the same route simultaneously. The therapeutic effects of BM-MSCs-RLX+GFP were then investigated in a high salt (HS; 2% NaCI)-induced model of hypertension. Flow cytometry revealed a transduc...
Introduction: Evidence suggests that hypertension involves underlying inflammation, however wheth... more Introduction: Evidence suggests that hypertension involves underlying inflammation, however whether atherosclerosis - a chronic inflammatory condition - can cause hypertension is unknown. We tested whether blood pressure (BP) is higher in high-fat fed ApoE-/- vs chow-fed wild-type (WT) mice, and whether advanced atherosclerosis is associated with systemic and end-organ inflammation, oxidative stress and endothelial dysfunction. Methods: Male ApoE-/- and WT mice were placed on high fat and chow diets, respectively, from 5-56 weeks. To clarify the effects of ageing alone, aged WT mice were compared to young chow-fed WT mice (8-12 week old). We measured systolic BP, plasma cytokine levels, mRNA expression of inflammatory markers, vascular superoxide and endothelial function. Results: There was no difference in BP of aged ApoE-/- (104.2 ± 2.9 mmHg) and age-matched WT mice (113.2 ± 1.3 mmHg) (n=13-18, P>0.05). However, plasma IL-6, TNF-α and IFN-γ were elevated in ApoE-/- by more than...
Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological... more Introduction: Hypertension in the obese is a rapidly increasing problem. It has both neurological and immunological antecedents, however the relationship between these is unclear. Methods and Results: Here we used a mouse model of diet-induced obesity, pressure myography, immunohistochemistry, flow cytometry and radiotelemtry to demonstrate that immune cells, located within the vascular adventitia of arteries important for controlling blood pressure, produce nerve growth factor (NGF) and rapidly initiate growth of the perivascular sympathetic nerve plexus; which increases vascular tone and thereby elevates blood pressure. We also show that Rag1-/- mice lacking T and B cells do not develop hypertension when obesity is induced with a high fat diet (HFD) and show that their sympathetic innervation and vascular tone remains normal, suggesting that neurogenic hypertension in the obese likely occurs independently of weight gain. Furthermore, we show that the adoptive transfer of wildtype ...
Inflammasomes are a family of interleukin-1 processing complexes and master regulators of inflamm... more Inflammasomes are a family of interleukin-1 processing complexes and master regulators of inflammation. We recently showed that both one-kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)- and angiotensin II-dependent hypertension in mice are associated with elevated expression of the NLRP3 inflammasome in the kidneys. Moreover, genetic deficiency of a key subunit critical for NLRP3 inflammasome activity protected mice against renal inflammation and chronic pressor responses associated with these models. As a step towards translation of these findings into new therapies, here we investigated whether a highly specific small-molecule inhibitor of NLRP3 inflammasome activity, MCC950, similarly reduces the deleterious effects of 1K/DOCA/salt on blood pressure (BP) and renal function. Male C57BL6/J mice were implanted with telemetry probes for continuous recording of BP (mean arterial (MAP), systolic, diastolic) and heart rate (HR), or placed in metabolic cages for 24 h urine collect...
Background and PurposeOxidative stress and fibrosis are hallmarks of cardiomyopathy‐induced heart... more Background and PurposeOxidative stress and fibrosis are hallmarks of cardiomyopathy‐induced heart failure yet are not effectively targeted by current frontline therapies. Here, the therapeutic effects of the anti‐oxidant, N‐acetylcysteine (NAC), were compared and combined with an acute heart failure drug with established anti‐fibrotic effects, serelaxin (RLX), in a murine model of cardiomyopathy.Experimental ApproachAdult male 129sv mice were subjected to repeated isoprenaline (25 mg·kg−1)‐induced cardiac injury for five consecutive days and then left to undergo fibrotic healing until Day 14. Subgroups of isoprenaline‐injured mice were treated with RLX (0.5 mg·kg−1·day−1), NAC (25 mg·kg−1·day−1) or both combined, given subcutaneously via osmotic minipumps from Day 7 to 14. Control mice received saline instead of isoprenaline.Key ResultsIsoprenaline‐injured mice showed increased left ventricular (LV) inflammation (~5‐fold), oxidative stress (~1–2.5‐fold), cardiomyocyte hypertrophy (~...
Interleukin-18 (IL-18) is a pro-inflammatory cytokine released downstream of inflammasome activat... more Interleukin-18 (IL-18) is a pro-inflammatory cytokine released downstream of inflammasome activation that is elevated in hypertensive patients. However, whether IL-18 plays a causal role in hyperte...
IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood p... more IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood pressure and end-organ damage is unknown. We examined the role of IL-18 in the development of renal inflammation and injury in a mouse model of low-renin hypertension. Hypertension was induced in male C57BL6/J (WT) and IL-18−/−mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% drinking saline (1K/DOCA/salt). Normotensive controls received uninephrectomy and placebo (1K/placebo). Blood pressure was measured via tail cuff or radiotelemetry. After 21 days, kidneys were harvested for (immuno)histochemical, quantitative-PCR and flow cytometric analyses of fibrosis, inflammation, and immune cell infiltration. 1K/DOCA/salt-treated WT mice developed hypertension, renal fibrosis, upregulation of proinflammatory genes, and accumulation of CD3+T cells in the kidneys. They also displayed increased expression of IL-18 on tubular epithelial cells. IL-18−/−mice were profoundly...
Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow ... more Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 μg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.
Background and PurposeFibrosis is a hallmark of chronic kidney disease (CKD) that significantly c... more Background and PurposeFibrosis is a hallmark of chronic kidney disease (CKD) that significantly contributes to renal dysfunction, and impairs the efficacy of stem cell‐based therapies. This study determined whether combining bone marrow‐derived mesenchymal stem cells (BM‐MSCs) with the renoprotective effects of recombinant human relaxin (serelaxin) could therapeutically reduce renal fibrosis in mice with one kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)‐induced hypertension, compared with the effects of the ACE inhibitor, perindopril.Experimental ApproachAdult male C57BL/6 mice were uni‐nephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt) for 21 days. Control mice were uni‐nephrectomised but received water over the same time period. Sub‐groups of 1K/DOCA/salt‐injured mice (n = 5–8 per group) were treated with either serelaxin (0.5 mg·kg−1·day−1) or BM‐MSCs (1 × 106 per mouse) alone; both treatments combined (with 0.5 × 106 or 1 × 106 BM...
Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapie... more Fibrosis significantly impairs the viability and therapeutic efficacy of stem cell-based therapies. To overcome this, we demonstrated that the anti-fibrotic drug, serelaxin (recombinant human relaxin; RLX), could prevent renal fibrosis progression in a unilateral ureteric obstruction (UUO)-induced murine model of interstitial renal disease and improve the ability of injected human bone marrow-derived mesenchymal stem cells (BM-MSCs) to attenuate renal injury. To advance the therapeutic potential of this combination therapy, we have now investigated its effects as an intervention therapy in the UUO model, and in a uninephrectomised/deoxycorticosterone acetate/drinking saline water (1K/DOCA/salt) model of hypertension. 7-8 week old male C57B6J mice were subjected to UUO or 1K/DOCA/salt (2.4mg/day, s.c; 21 days). Sub-groups of injured mice (n=6/group) were administered RLX (0.5mg/kg/day; s.c) alone, BM-MSCs (1x10 6 /mouse; i.v) alone or both combined 3-days post-UUO or 14 days post-1K/...
To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal infl... more To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension. Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys. By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P<0.0001). The intervention with anakinra reduced BP in 1K/DOCA/salt-treated mice by ∼20mmHg (n=16, P<0.05)...
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Papers by Dorota Ferens