#1

The challenge of restoring or increasing the elastic fiber content in aged patients or animals (or in pathological conditions) is of particular importance and consists in stimulating the production of the elastic fiber components at a time when the synthesis of several of them is very low.

Tropoelastin, the soluble precursor of elastin, and other elastic fiber components are synthesized in the skin by dermal fibroblasts and in vessels by medial VSMCs. The synthesis of tropoelastin is regulated by numerous chemokines and growth factors and also by potassium (K+) and calcium (Ca2+) ions. Indeed, K+ excess in the extracellular medium induces the depolarization of the plasma membrane, which causes the opening of voltage-dependent calcium channels (CaV) and leads to an increase in [Ca2+]i and a decrease in elastin synthesis. Such a decrease in elastin synthesis was also shown in VSMCs incubated with the calcium ionophore A23187, which triggers Ca2+ influx and [Ca2+]i elevation. On the contrary, K+ efflux from cells leads to an increase in elastin synthesis due to membrane hyperpolarization and CaV closing. This has been shown in experiments using the ATP-dependent K+ (KATP) channel opener minoxidil, a potent arterial vasodilator which has long been used to treat hypertension. Minoxidil-induced K+ efflux leads to enhancement of elastin synthesis in cultured VSMCs and skin fibroblasts. In vivo studies have also demonstrated that the elastin content and/or thickness of elastic lamellae are increased by minoxidil treatment in several animal models: Spontaneously Hypertensive Rats (SHR). Chronic treatment of mice with minoxidil also results in improvements of the arterial mechanics and cerebral perfusion and, at least in aged animals, in neosynthesis of elastic fibers and protection of pre-existing elastic fibers. Other KATP channel openers, like diazoxide or nicorandil, exert an effect on in vivo elastin synthesis similar to those induced by minoxidil, while pinacidil and some cromakalim or diazoxide derivatives have also been shown to induce elastin production by cultured VSMCs in rats. However, treatment of animals with other antihypertensive drugs, either angiotensin-II type 1 receptor blocker, beta blocker or calcium channel blocker, does not modify the elastin content in mouse arteries.

Numerous cytokines which inhibit tropoelastin transcription act through the activation of the Ras/MEK/ERK signaling pathway. The increase in [Ca2+]i in VSMCs also stimulates ERK1/2 phosphorylation. The inhibition of this signaling pathway could thus be a strategy to increase the elastin content in arteries. Actually, by performing in vitro and in vivo experiments in rats, it has been demonstrated that inhibition of the ERK1/2 phosphorylation leads to increases in elastin synthesis by VSMCs and elastin content in the aorta. The importance of this pathway in elastin metabolism has been confirmed by the demonstration that cortistatin reduces elastin degradation, MMP-2 and -9 expressions and aneurysm progression through the inhibition of the ERK1/2 signaling pathways. Of importance, it has also previously been shown that ERK1/2 signaling is involved in elastin peptide signaling through the elastin receptor complex (ERC). The general impact of the pharmacological treatments modulating the KATP channels-Ca2+ channels-ERK1/2 pathways described above is illustrated in Fig. 1.

Application of potassium channel openers to VSMCs leads to cell membrane hyperpolarization and calcium channel blockade. This induces a drop in intracellular Ca2+ concentration and inhibition of the ERK 1/2 pathway, resulting in the activation of the production of mRNAs involved in elastic fiber synthesis (tropoelastin, TE; fibulin-5, FBLN-5; lysyl-oxidase, LOX; fibrillin-1, FBN-1; …) and secretion of the corresponding proteins. Treatment of the cells with an inhibitor of ERK 1/2 phosphorylation produces the same effect regarding elastin. Elastic fiber neosynthesis then precedes their integration/aggregation into pre-existing elastic fibers and elastic lamellae. Regarding the effect of minoxidil, a limitation of elastic fiber ruptures and AGE formation is also observed.

MEK inhibitors have also been used in order to stimulate elastin synthesis by cells isolated from patients with Costello syndrome, a developmental disorder characterized by hyper-activation of the mitogenic Ras-Raf-MEK/ERK pathway and inhibition of elastogenesis. The treatment of dermal fibroblasts derived from these patients with the MEK inhibitor PD98059 leads to the concomitant inhibition of cell proliferation and recovery of elastin production.

Completely different therapeutic strategies have also been tested to stimulate elastin synthesis using in vitro or in vivo models. Cenizo and colleagues have used a dill extract to induce LOXL gene expression in dermal fibroblasts, resulting in elastin content increase in dermal and skin equivalents as well as skin elasticity elevation in vivo in humans.

In addition, based on a previous work which has shown that microRNA (miR)-29 mimics downregulate the expression of elastin (ELN) as well as parts of collagen type I (COL1A1) and collagen type III (COL3A1) genes, the team of William C. Sessa has demonstrated that inhibition of miR-29a can increase ELN expression in human cells (skin fibroblasts, VSMCs, bioengineered vessels) and in cells from patients with ELN haploinsufficiencies (supravalvular aortic stenosis or Williams-Beuren syndrome). Using the same in vitro models, it was also demonstrated that engineered zinc-finger protein transcription factors that target the ELN gene can also be used to stimulate the expression of elastin. However, these potential therapeutic molecules have not been tested in vivo yet and need additional investigations for validation of their therapeutic potential.

https://sci-hub.se/downloads/2019-10-05/48/fhayli2019.pdf?download=true

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#2

Background: Development of albuminuria and arterial stiffness in Munich Wistar Frömter (MWF) rats, a model of chronic kidney disease, is related to alterations in extracellular matrix, increased oxidative stress, and endothelial dysfunction. Finerenone (FIN), a novel, nonsteroidal, potent, and selective mineralocorticoid receptor antagonist, improves endothelial dysfunction through enhancing nitric oxide (NO) bioavailability and decreasing superoxide anion levels due to an upregulation in vascular and renal superoxide dismutase activity. We hypothesize that FIN reduces arterial stiffness in this model associated to the reduction in albuminuria and matrix metalloproteinase (MMP)-2/9 activity.

Methods: Twelve-week-old MWF rats with established albuminuria and age-matched normoalbuminuric Wistar (W) rats were treated with FIN (10 mg/kg/day, once-daily oral gavage) or with vehicle (control, C) for 4 weeks.

Results: Arterial stiffness was significantly higher in mesenteric arteries (MA) of MWF-C as compared to W-C. FIN treatment significantly lowered β-index, a measure of intrinsic stiffness independent of geometry, in MWF (βMWF-FIN = 7.7 ± 0.4 vs. βMWF-C = 9.2 ± 0.5, p < 0.05) positively correlating with urinary albumin excretion. Elastin fenestrae area in the internal elastic lamina of MA from MWF-FIN was significantly larger (+377%, p < 0.05). FIN increased plasma pro-MMP-2 and decreased plasma MMP-2 and MMP-9 activities, correlating with reductions in β-index. MA from MWF-FIN exhibited higher NO bioavailability and reduced superoxide anion levels compared to MWF-C.

Conclusion: FIN treatment reduces intrinsic arterial stiffness in MA from MWF rats associated with changes in elastin organization, normalization of MMP-2 and MMP-9 activities, and reduction of oxidative stress. Moreover, reduction of arterial stiffness correlates with reduction in albuminuria.

Introduction: Several studies show the association between albuminuria and arterial stiffness in hypertensive and diabetic patients, as well as in the general population. In a genetic rat model with spontaneous albuminuria, the Munich Wistar Frömter (MWF) rat, albuminuria is associated to an increased arterial stiffness due to a reduction in elastin content.

Hypothesis: Finerenone, a next-generation, oral, selective, and non-steroidal mineralocorticoid receptor antagonist, reduces arterial stiffness.

Methods: Twelve week-old MWF and aged-matched normoalbuminuric Wistar (W) rats were treated for 4-week with finerenone (F, 10 mg/kg/day). We analysed i) structural and mechanical parameters in third order mesenteric resistance arteries (MRA) by pressure myography, and ii) the structure and organization of elastin in vascular elastic laminae (EL) by confocal microscopy. Arterial stiffness was estimated by determination of β-values, which reflect intrinsic stiffness of the wall material independently of the geometry as obtained from stress-strain relationship.

Results: Treatment with F significantly reduced SBP but no DBP in MWF-F. No effect was observed on pulse wave velocity. MRA from MWF-C did not exhibit important structural changes (vessel diameter, lumen diameter, wall thickness, wall to lumen ratio and cross sectional area) compared to W-C. However, we observed a significant increase in arterial stiffness in MWF-C as assessed by β-values (βW-C:6.4±0.3 vs βMWF-C:9.2±0.5, p<0.05). F significantly reduced β-values in MWF (-20%, p<0.05). Elastin content in both the external and the internal EL was similar between strains. The area of the fenestra in the internal EL was lower in MWF-C compared to W-C (W-C:966±192μm2 vs MWF-C:518±108 μm2; p<0.05). Treatment with F led to a striking increase in increased fenestrae area in MWF (+377%, p<0.05).

Conclusion: Treatment with F significantly reduces both BP and arterial stiffness in a rat model with albuminuria. The reduction of arterial stiffness is related to changes in elastin organization and, more precisely, to the enlargement of fenestrae area. The latter may also allow a better diffusion from endothelial factors to the underlying smooth muscle layer thereby influencing vascular function.

https://www.ahajournals.org/doi/10.1161/circ.136.suppl_1.13793

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How to Reverse Skin Aging
#3

Objective:

Elastin gene deletion or mutation leads to arterial stenoses due to vascular smooth muscle cell (SMC) proliferation. Human induced pluripotent stem cells–derived SMCs can model the elastin insufficiency phenotype in vitro but show only partial rescue with rapamycin. Our objective was to identify drug candidates with superior efficacy in rescuing the SMC phenotype in elastin insufficiency patients.

Approach and Results:

SMCs generated from induced pluripotent stem cells from 5 elastin insufficiency patients with severe recurrent vascular stenoses (3 Williams syndrome and 2 elastin mutations) were phenotypically immature, hyperproliferative, poorly responsive to endothelin, and exerted reduced tension in 3-dimensional smooth muscle biowires. Elastin mRNA and protein were reduced in SMCs from patients compared to healthy control SMCs. Fourteen drug candidates were tested on patient SMCs. Of the mammalian target of rapamycin inhibitors studied, everolimus restored differentiation, rescued proliferation, and improved endothelin-induced calcium flux in all patient SMCs except one Williams syndrome. Of the calcium channel blockers, verapamil increased SMC differentiation and reduced proliferation in Williams syndrome patient cells but not in elastin mutation patients and had no effect on endothelin response. Combination treatment with everolimus and verapamil was not superior to everolimus alone. Other drug candidates had limited efficacy.

Conclusions:

Everolimus caused the most consistent improvement in SMC differentiation, proliferation and in SMC function in patients with both syndromic and nonsyndromic elastin insufficiency, and offers the best candidate for drug repurposing for treatment of elastin insufficiency associated vasculopathy.

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#4

Calcium blockers most likely decrease the levels of elastin in your body.

Aims: Abdominal aortic aneurysms are characterised by changes in the extracellular matrix of the arterial media, in particular a reduction in elastin concentration. These changes are mediated by increased levels of endogenous matrix metalloproteinases (MMPs). Recently, calcium channel blockers have been shown to increase the proteolytic activity of MMP-2 secreted by vascular smooth muscle cells. It may therefore by hypothesised that calcium antagonists may potentiate the activity of MMPs in aneurysmal disease and thus accelerate AAA expansion. In this study, the ability of amlodipine–a calcium antagonist–to influence elastin degradation, was assessed in a previously described model of aneurysmal disease.

Methods: Porcine aortic segments (n = 8) were pre-incubated in exogenous pancreatic elastase for 24 h prior to culture in standard conditions for 6 days with 10 and 100 micrograms/l amlodipine. Control segments were cultured both with and without amlodipine and without elastase. At the termination of culture MMPs were extracted from the tissue and quantified by a combination of substrate gel enzymography and immunoblotting. The volume fractions of elastin and collagen were determined by stereological analysis of EVG stained sections.

Results: Gel enzymography demonstrated significantly increased MMP-9 activity in the amlodipine treated segments, median 4.218 vs. 2.809 arbitrary units (p < 0.01) and this elevated activity was reflected in a significant destruction of medial elastin 27.0 vs. 40.5% (p < 0.05).

Conclusion: Therapeutic ranges of amlodipine significantly enhanced elastin degradation and potentiated MMP-9 activity within the aortic organ cultures.

Elastin synthesis in cultured smooth muscle cells was inhibited by one fourth in the presence of 0.1 M K+ in the medium. The degree of inhibition paralleled the decrease in the steady-state levels of elastin mRNA. The inhibition of elastin synthesis was blocked by addition of 1 microM nifedipine, a Ca2+ antagonist. Comparable inhibition of elastin synthesis was observed by addition of A23187, a Ca2+ ionophore. In contrast, collagen synthesis and thymidine uptake were stimulated threefold and twofold respectively in the presence of 0.1 M K+ with a concomitant increase in collagen mRNA. The stimulation of collagen synthesis was also blocked by nifedipine. These results indicate that K+ modulates elastin and collagen synthesis and their gene expression reciprocally, and these effects are mediated by Ca2+ influx. Thus K+ exerts profound effects on the composition of extracellular matrices in aorta.

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#5

Our study was underpowered to detect a decrease of IMT and arterial stiffness or distensibility in CCA or in more muscular vessels such as humeral artery in children with WBS treated by minoxidil. Surprisingly, our results indicate that minoxidil tend to slightly increase IMT, and decrease distensibility. Exploring the renin angiotensin system [14], elastin gene transcription and its signaling pathway [45] and potential new biomarkers [47], we are hopeful to better explain how minoxidil influences biologically the elastogenesis in patients with WBS. Differences between groups in vessel wall thickening of 0.02 mm was very small. The Bland & Altman graphs (Additional file 2: Figures) show that the difference between measure 1 and 2 decreases after the first reading. The sensitivity analysis also suggests that the measurement covariates do not influence the treatment effect.

We also explored the potential interaction between the diameter of the carotid artery or the mean ambulatory blood pressure and the treatment effect on the IMT during the 12-month follow-up. Our results show, a positive correlation in the minoxidil group between the diastolic (r = 0.08) and systolic (r = 0,42) carotid diameter changes with IMT change. These carotid diameter changes are also positively correlated with IMT change in the placebo group (r diastolic = 0.61 and r systolic = 0.75) with no apparent interaction between carotid diameters changes and the treatment group (p interaction = 0,11 for diastolic and p = 0,07 for systolic). The mean ambulatory diastolic and systolic BP changes were positively correlated in the minoxidil (r sys and diast = 0.20), but negatively correlated with IMT change in the placebo group (r syt = -0.09, r diast = -0.45).

The interaction between mean ambulatory BP and the treatment group was statistically significant (p = 0.018) indicating that when the blood pressure increase, IMT decreases in the placebo group but still increase paradoxically in the minoxidil group.

We assumed that minoxidil could be potentially beneficial in children because elastin is synthesized in early life. We set the duration of the treatment on one-year to make sure that the elastogenesis could be stimulated. There is no direct evidence, however, on the time needed to stimulate elastogenesis in children. Animal data have shown that two months treatment with high dose of potassium channel openers seems enough to stimulate elastogenesis [14, 45]. The low dose of minoxidil used in children compared with the doses used in animals and the shorter duration of the exposure relative to the life span of children could explain our inconclusive results.

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#6

We ask the question “Are Arao‐Perkins elastin bodies, containing abnormally deposited elastin, preventing the reversal of hair follicle miniaturisation in female and male pattern hair loss?” In order to explore this, we propose that scalp tissue from males and females with pattern hair loss is stained using both the histochemical stains as used originally, but also complimented with antibody staining for both elastin and collagen. The lysyl oxidase (LOX) family encodes copper‐dependent amine oxidases that are important in determining the tensile strength and structural integrity of connective tissues, including skin, by catalysing the crosslinking of elastin or collagen. 53 LOX functions in the organisation of the extracellular matrix in the ovary under the influence of hormonal activity 54 , 55 and LOX with TGFβ regulates elastin gene expression in dermal fibroblasts. 56 We also consider other fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF) and kidney disease that also involve alteration in elastin deposition under the influence of TGFβ and LOX. IPF is associated with elevated elastin‐collagen crosslinking and activation of myofibroblasts—a cell type that forms the follicle connective tissue sheath (CTS). The extracellular matrix exhibits a loss of elasticity and in the lung, this equated to loss of tissue function. 57 Kidney disease fibrosis is also associated with greater crosslinking of the collagen and elastin fibres due to elevation of LOX. Techniques used in renal disease pathology include elastin imaging 58 so comparison of the follicle in AGA and kidney would be a worthy exercise, not least as these two organs have already been compared recently in this journal.

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#7

Haploinsufficiency of elastin in patients with WBS leads, in more than half of cases, to development of SVAS and hypertension. The discovery of molecules able to enhance elastin synthesis would be of interest to treat this condition. In the present study, we demonstrated that an increase in [Ca2+]i decreases (1) the steady-state level of mRNAs encoding for proteins composing elastic fibers in vitro, (2) both elastin gene transcription and its mRNA stability, and (3) elastin gene transcription via activation of the ERK1/2-AP1 signaling pathway. Moreover, we have shown that inhibition of ERK1/2 phosphorylation increases (1) elastin gene transcription in vitro without affecting the steady-state level of mRNAs encoding for other proteins composing elastic fibers and (2) elastic fiber thickness and aortic insoluble elastin content in the BN rat in vivo, without functional or morphological cardiovascular alterations. Silencing of c-fos and c-jun, two AP1 transcription factors that are downstream to ERK1/2, increases elastin synthesis in vitro; thus, it would be of interest to further study the effect of silencing AP1 transcription factors on elastogenesis in vivo. It would be also of interest to treat elastin-deficient or haploinsufficient mice with elastogenic molecules, such as U0126, to analyze the effect of elastin neosynthesis on the development of aortic stenosis and hypertension. Inhibitors of ERK1/2 phosphorylation could be used in a clinical trial for treating young patients with WBS.

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#8

Since MMP12 is an ECM protein that primarily degrades elastin, we hypothesized that MMP12 deficiency might also lead to improved elasticity and biomechanical properties of the atherosclerotic arterial wall. We assessed the axial and circumferential stretch of thoracic aortas, which revealed a marked improvement of aortic stiffness in DKO mice (Fig. 4C), with a significantly softer axial mechanical behavior at axial stretches > 1.2. Given that the axial inversion stretches of the mouse aortas studied ranged from 1.3 to 1.75, differences in axial mechanical properties between DKO and LdlrKO mice at axial prestretches in vivo are to be expected. The circumferential stretch of the LdlrKO aortas (1.17 ± 0.01) was significantly lower than the corresponding stretch of the DKO mice (1.39 ± 0.09) (Fig. 4D), indicating a softer mechanical behavior of DKO aortas also in the circumferential direction. In line, electron microscopy revealed only remnants of elastic fibers that appeared to be replaced by collagen in aortas from LdlrKO mice (Fig. 4E), whereas the elastic fibers remained intact in the aortas from DKO animals (Fig. 4F). Overall, the mechanical behavior in the axial and circumferential directions and the electron micrographs suggest that genetic deletion of MMP12 results in better preservation of elastic fibers, which translates into diminished arterial stiffness of the atherosclerotic vessel.

We conclude that the genetic deletion of MMP12 in a mouse model mimicking CMDs in humans leads to altered immune cell composition and improvements in systemic inflammation, insulin sensitivity, and atherosclerotic development, supported by beneficial changes in the proteome signature of WAT and aortas. In humans, circulating MMP12 levels are weakly linked to MS and indicators of CMDs. Further studies are needed to establish the association between tissue MMP12 bioavailability and CMDs.

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#9

Is the calcium ion vs potassium ion issue relevant to Marfan’s? I was not aware of the Minoxidil concern for Ehlers Danlos sufferers. Apparently there are a bunch of meds that affect connective tissue. Some help, and some are problematic. I am hopeful that Rapamycin or Everolimus are beneficial for Marfan’s.

#10

Maybe.

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome and related inherited thoracic aortic aneurysm (TAA) syndromes for prophylaxis against TAA progression, despite limited evidence for their efficacy in these disorders. Unexpectedly, Marfan mice treated with CCBs showed accelerated TAA expansion, rupture and premature lethality. In collaboration with other members of the GenTAC consortium, we conducted a case-control study to assess the effect of CCBs in humans with Marfan syndrome and related TAA conditions. These primarily included Loeys-Dietz, Turner and Ehlers-Danlos syndromes, familial thoracic aortic aneurysm, and bicuspid aortic valve with aneurysm. Marfan patients with native aortic roots at the time of enrollment who received CCBs (compared to other antihypertensive agents) had an increased risk of aortic dissection (odds ratio (OR) 12.5, p<0.05). Strong trends were maintained after correction for blood pressure (OR 12.7, p=0.06) or aortic root size (OR 11.2, p=0.08) at enrollment. A more profound effect of CCBs on outcome could be masked by the practice of intervening with aortic surgery when the aorta reaches a dimension that confers risk for dissection. In keeping with this hypothesis, we found that CCB-treated Marfan patients had an increased risk of aortic surgery (OR 5.5, p<0.001) when compared to patients on other antihypertensive agents, which remained significant when corrected for either blood pressure (OR 5.4, p<0.001) or aortic size (OR 5.0, p<0.01) at enrollment. For patients with other forms of TAA and native aortic roots at the time of enrollment (n=1,819), there was suggestion of an increased risk for aortic dissection in those who had taken CCBs, although this did not reach statistical significance (OR 4.7, p=0.26). This was most likely secondary to prophylactic surgical intervention, given that these patients again had an increased risk of aortic surgery (OR 2.4, p=0.004), which remained significant when corrected for either blood pressure (OR 2.2, p=0.016) or aortic size (OR 2.2, p=0.017) at enrollment. These data indicate that CCB treatment exacerbates aortic disease in Marfan syndrome and related TAA conditions, and suggests that CCBs should be used with caution in these patient populations.

https://www.ahajournals.org/doi/10.1161/circ.130.suppl_2.18126

#11

It seems like calcium channel blockers may be detrimental and beneficial at the same time. They may impair connective tissue repair, but they lower blood pressure effectively. Beta blockers are ARB are prescribed for Marfan’s frequently. The thinking here is limited if just think of the heart as a mechanical pump, and the aorta as a hose. Lowered BP is helpful, but the real fix is getting connective tissue to repair correctly. I would gamble that Rapamycin will help, but it could make things worse.