Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/− and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/− aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/− mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/− VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/− aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/− aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/− mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.</p

Ercc1 DNA repair deficiency results in vascular aging characterized by VSMC phenotype switching, ECM remodeling, and an increased stress response

Cardiovascular diseases are the number one cause of death globally. The most important determinant of cardiovascular health is a person's age. Aging results in structural changes and functional decline of the cardiovascular system. DNA damage is an important contributor to the aging process, and mice with a DNA repair defect caused by Ercc1 deficiency display hypertension, vascular stiffening, and loss of vasomotor control. To determine the underlying cause, we compared important hallmarks of vascular aging in aortas of both Ercc1Δ/− and age-matched wildtype mice. Additionally, we investigated vascular aging in 104 week old wildtype mice. Ercc1Δ/− aortas displayed arterial thickening, a loss of cells, and a discontinuous endothelial layer. Aortas of 24 week old Ercc1Δ/− mice showed phenotypical switching of vascular smooth muscle cells (VSMCs), characterized by a decrease in contractile markers and a decrease in synthetic markers at the RNA level. As well as an increase in osteogenic markers, microcalcification, and an increase in markers for damage induced stress response. This suggests that Ercc1Δ/− VSMCs undergo a stress-induced contractile-to-osteogenic phenotype switch. Ercc1Δ/− aortas showed increased MMP activity, elastin fragmentation, and proteoglycan deposition, characteristic of vascular aging and indicative of age-related extracellular matrix remodeling. The 104 week old WT mice showed loss of cells, VSMC dedifferentiation, and senescence. In conclusion, Ercc1Δ/− aortas rapidly display many characteristics of vascular aging, and thus the Ercc1Δ/− mouse is an excellent model to evaluate drugs that prevent vascular aging in a short time span at the functional, histological, and cellular level.</p

Folate Receptor–Targeted Single-Photon Emission Computed Tomography/Computed Tomography to Detect Activated Macrophages in Atherosclerosis: Can It Distinguish Vulnerable from Stable Atherosclerotic Plaques?

The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical (111In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E 7 mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed 111In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of 111In-EC0800 in the stable plaque normalized to plaque volume. Our data show that 111In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis.ISSN:1535-3508ISSN:1536-012

Folate Receptor–Targeted Single-Photon Emission Computed Tomography/Computed Tomography to Detect Activated Macrophages in Atherosclerosis: Can It Distinguish Vulnerable from Stable Atherosclerotic Plaques?

The need for noninvasive imaging to distinguish stable from vulnerable atherosclerotic plaques is evident. Activated macrophages play a role in atherosclerosis and express folate receptor folate receptor β (FR-β). The feasibility of folate targeting to detect atherosclerosis was demonstrated in human and mouse plaques, and it was suggested that molecular imaging of FR-β through folate conjugates might be a specific marker for plaque vulnerability. However, these studies did not allow differentiation between stable and vulnerable atherosclerotic plaques. We investigated the feasibility of a folate-based radiopharmaceutical ( 111 In-EC0800) with high-resolution animal single-photon emission computed tomography/computed tomography (SPECT/CT) to differentiate between stable and vulnerable atherosclerotic plaques in apolipoprotein E 7 mice in which we can induce plaques with the characteristics of stable and vulnerable plaques by placing a flow-modifying cast around the common carotid artery. Both plaques showed 111 In-EC0800 uptake, with higher uptake in the vulnerable plaque. However, the vulnerable plaque was larger than the stable plaque. Therefore, we determined tracer uptake per plaque volume and demonstrated higher accumulation of 111 In-EC0800 in the stable plaque normalized to plaque volume. Our data show that 111 In-EC0800 is not a clear-cut marker for the detection of vulnerable plaques but detects both stable and vulnerable atherosclerotic plaques in a mouse model of atherosclerosis

In vivo renin activity imaging in the kidney of progeroid Ercc1 mutant mice

Changes in the renin–angiotensin system, known for its critical role in the regulation of blood pressure and sodium homeostasis, may contribute to aging and age-related diseases. While the renin–angiotensin system is suppressed during aging, little is known about its regulation and activity within tissues. However, this knowledge is required to successively treat or prevent renal disease in the elderly. Ercc1 is involved in important DNA repair pathways, and when mutated causes accelerated aging phenotypes in humans and mice. In this study, we hypothesized that unrepaired DNA damage contributes to accelerated kidney failure. We tested the use of the renin-activatable near-infrared fluorescent probe ReninSense680™ in progeroid Ercc1d/− mice and compared renin activity levels in vivo to wild-type mice. First, we validated the specificity of the probe by detecting increased intrarenal activity after losartan treatment and the virtual absence of fluorescence in renin knock-out mice. Second, age-related kidney pathology, tubular anisokaryosis, glomerulosclerosis and increased apoptosis were confirmed in the kidneys of 24-week-old Ercc1d/− mice, while initial renal development was normal. Next, we examined the in vivo renin activity in these Ercc1d/− mice. Interestingly, increased intrarenal renin activity was detected by ReninSense in Ercc1d/− compared to WT mice, while their plasma renin concentrations were lower. Hence, this study demonstrates that intrarenal RAS activity does not necessarily run in parallel with circulating renin in the aging mouse. In addition, our study supports the use of this probe for longitudinal imaging of altered RAS signaling in aging

Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model (Ercc1Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser1177-eNOS were compromised in Ercc1Δ/- DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1Δ/- mice. Ercc1Δ/- mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective

Extracellular Matrix Defects in Aneurysmal Fibulin-4 Mice Predispose to Lung Emphysema

<div><p>Background</p><p>In this study we set out to investigate the clinically observed relationship between chronic obstructive pulmonary disease (COPD) and aortic aneurysms. We tested the hypothesis that an inherited deficiency of connective tissue might play a role in the combined development of pulmonary emphysema and vascular disease.</p><p>Methods</p><p>We first determined the prevalence of chronic obstructive pulmonary disease in a clinical cohort of aortic aneurysms patients and arterial occlusive disease patients. Subsequently, we used a combined approach comprising pathological, functional, molecular imaging, immunological and gene expression analysis to reveal the sequence of events that culminates in pulmonary emphysema in aneurysmal Fibulin-4 deficient (Fibulin-4^R) mice.</p><p>Results</p><p>Here we show that COPD is significantly more prevalent in aneurysm patients compared to arterial occlusive disease patients, independent of smoking, other clinical risk factors and inflammation. In addition, we demonstrate that aneurysmal Fibulin-4^R/R mice display severe developmental lung emphysema, whereas Fibulin-4^+/R mice acquire alveolar breakdown with age and upon infectious stress. This vicious circle is further exacerbated by the diminished antiprotease capacity of the lungs and ultimately results in the development of pulmonary emphysema.</p><p>Conclusions</p><p>Our experimental data identify genetic susceptibility to extracellular matrix degradation and secondary inflammation as the common mechanisms in both COPD and aneurysm formation.</p></div

Enlarged alveolar airspaces in lungs of Fibulin-4 knockdown mice.

<p>(A) Expression levels of Fibulin-4 in lungs isolated from newborn (n = 4, n = 4, n = 3) and adult (n = 4, n = 4, n = 4) Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R mice relative to Fibulin-4^+/+ lungs (**p<0.01). (B) Mean peak inspiratory flow (PIF) and peak expiratory flow (PEF) values for Fibulin-4^+/+ (n = 4), Fibulin-4^+/R (n = 4) and Fibulin-4^R/R mice (observed for n = 4, but two animals died during the procedure) at 3-minute intervals. After a 9 minute adaptation period (the first three time intervals), PIF follows similar trends in Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R mice, while Fibulin-4^R/R mice show a decrease in PEF. (C) HE stained sections of formalin fixed lungs of male mice. Enlarged alveolar airspaces are observed in Fibulin-4^+/R (middle, n = 3) and Fibulin-4^R/R lungs (right, n = 3), with the latter being more pronounced, compared to Fibulin-4^+/+ (n = 3). Enlarged alveolar airspaces are already present in Fibulin-4^R/R newborn lungs (n = 3), while lungs of Fibulin-4^+/R littermates (n = 5) show no difference compared to Fibulin-4^+/+ lungs (n = 4). Scale bar 100 µm. Magnification 10x. (D) D₂ quantification (see methods and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106054#pone.0106054.s001" target="_blank">Figure S1</a> for further explanation) of the alveolar airspaces revealed a significant difference between adult Fibulin-4^+/+ and Fibulin-4^+/R (*p<0.05) and between adult Fibulin-4^+/+ and Fibulin-4^R/R lungs (**p<0.01) as well as between newborn Fibulin-4^+/+ and Fibulin-4^R/R lungs (*p<0.05).</p

Increased inflammation in Fibulin-4^R/R lungs compared to Fibulin-4^+/+ and Fibulin-4^+/R lungs.

<p>(A) Flow cytometric analysis revealed more Gr1+ granulocytes and CD19+ B-cells in BAL samples from Fibulin-4^R/R (n = 4) compared to Fibulin-4^+/+ mice (n = 4, *p<0.05) and (B) increased numbers of F4/80 macrophages in Fibulin-4^R/R lungs (n = 4, *p<0.05). (C) HE stained sections from adult (n = 4, n = 4, n = 4) Fibulin-4^+/+, Fibulin-4^+/R and Fibulin-4^R/R lungs showing focal infiltrations around vessels and airways in Fibulin-4^R/R lungs (black arrows). (D) Staining for T-cells (CD3+) and (E) dendritic cells (CD11c+) points to the presence of inflammatory cells within the focal infiltrations. Magnification 20x. Scale bar 50 µm. (F) ELISA analysis showing increased IL-1β levels in Fibulin-4^R/R lungs (n = 4, *p<0.05).</p