Search

Mutations in the LMNA gene (encoding lamin A/C proteins) cause several human cardiac diseases, including dilated cardiomyopathies (LMNA-DCM). The main clinical risks in LMNA-DCM patients are sudden cardiac death and progressive left ventricular ejection fraction deterioration, and therefore most human and animal studies have sought to define the mechanisms through which LMNA mutations provoke cardiac alterations, with a particular focus on cardiomyocytes. To investigate if LMNA mutations also cause vascular alterations that might contribute to the etiopathogenesis of LMNA-DCM, we generated and characterized Lmnaflox/floxSM22αCre mice, which constitutively lack lamin A/C in vascular smooth muscle cells (VSMCs), cardiac fibroblasts, and cardiomyocytes. Like mice with whole body or cardiomyocyte-specific lamin A/C ablation, Lmnaflox/floxSM22αCre mice recapitulated the main hallmarks of human LMNA-DCM, including ventricular systolic dysfunction, cardiac conduction defects, cardiac fibrosis, and premature death. These alterations were associated with elevated expression of total and phosphorylated (active) Smad3 and cleaved (active) caspase 3 in the heart. Lmnaflox/floxSM22αCre mice also exhibited perivascular fibrosis in the coronary arteries and a switch of aortic VSMCs from the ‘contractile’ to the ‘synthetic’ phenotype. Ex vivo wire myography in isolated aortic rings revealed impaired maximum contraction capacity and an altered response to vasoconstrictor and vasodilator agents in Lmnaflox/floxSM22αCre mice. To our knowledge, our results provide the first evidence of phenotypic alterations in VSMCs that might contribute significantly to the pathophysiology of some forms of LMNA-DCM. Future work addressing the mechanisms underlying vascular defects in LMNA-DCM may open new therapeutic avenues for these diseases.

18 p.-6 fig.-1 tab., Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS., Work supported by Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) (MCIN/AEI/https://doi.org/10.13039/501100011033 grants SAF2016-79490-R and PID2019-108489RB-I00), with co-funding from Fondo Social Europeo (“El FSE invierte en tu futuro”), and a donation from Asociación Progeria Alexandra Peraut. Microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/https://doi.org/10.13039/501100011033. R.M.N was supported by the Ministerio de Educación, Cultura y Deporte (pre-doctoral contract FPU16/05027), and I.B. is supported by the Comunidad Autónoma de Madrid (grants 2017-T1/BMD-5247 and 2021-5A/BMD-20944), and the Ramón y Cajal contract (RYC2021-033805-I) funded by MCIN/AEI/10.13039/501100011033 and the European Union “NextGenerationEU”/PRTR. The CNIC is supported by the MCIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MCIN/AEI/https://doi.org/10.13039/501100011033).

We aimed to determine, in patients with type 1 diabetes (T1DM), the impact of excluding hyperglycemia as a criterion from the International Diabetes Federation (IDF) definition of the metabolic syndrome (MetS), both on its prevalence and on its association with micro and macrovascular complications and markers of subclinical inflammation.A cross-sectional design, including 280 patients with T1DM. We defined MetS by three different models: (i) the standard IDF criteria, (ii) a modification consisting of excluding of hyperglycemia as a criterion (modified IDF criteria) and (iii) a modification consisting in changing the hyperglycemia by insulin resistance (MetS + IR model) defined by the estimated glucose disposal rate. Microvascular complications and cardioautonomic neuropathy were assessed. We measured an inflammatory panel including high sensitivity C reactive protein, erythrocyte sedimentation rate, homocysteine, and fibrinogen concentrations.After excluding hyperglycemia, the prevalence of MetS was 6.4% (95%CI: 4.1 to 9.9) compared with 20.7% (95%CI: 16.3 to 25.8) using standard IDF criteria. After adjusting for duration of diabetes, all three MetS definitions increased the odds for having microvascular complications [OR: 6.012 (2.208-16.307) for modified definition; OR: 5.176 (2.555-10.486) for standard definition and [OR: 3.374 (1.649-8.456) for MetS+IR model]. However, the both modified IDF models for MetS showed better predictive performance than standard criteria for suffering from neuropathy, nephropathy, cardiovascular disease and were associated with markers of subclinical inflammation.The prevalence of MetS significantly varies as a function whether or not hyperglycemia is included as a diagnostic criterion. The subset of patients fulfilling the modified MetS definitions may reflect better the concept of metabolic syndrome in T1DM. These modified definitions were accompanied by a poorer metabolic control and lipid profile, showing the worse inflammatory biomarker profiles and higher odds for micro- and macrovascular complications. In patients with T1DM, the inclusion of insulin resistance instead of hyperglycemia as a criterion of MetS may be of interest in routine clinical practice.

Introduction Cardiovascular autonomic neuropathy (CAN) associates an abnormal circadian pattern in blood pressure (BP) regulation that might be aggravated by the coexistence of arterial stiffness. We aimed to evaluate the effect of arterial stiffness in the circadian rhythm of BP in patients with type 1 diabetes and CAN. Methods Cross-sectional study including 56 consecutive patients with type 1 diabetes and CAN, with ( n = 28) or without ( n = 24) arterial stiffness as defined by an ankle-brachial index above 1.2. CAN was diagnosed by BP and heart rate responses to active standing and cardiovascular autonomic reflex tests. Absence of nocturnal decrease in BP–“ non-dipping” pattern– was defined by a daytime to nighttime decrease in mean BP smaller than 10%. Results The study’s subjects mean age was 40 ± 11 years-old, their mean duration of diabetes was 22 ± 10 years, and their mean A1c was 7.9 ± 1.5%. A “ non-dipping” pattern was observed in 28 patients (54%) regardless of the presence or absence of arterial stiffness. Age, waist circumference, body mass index, and A1c, were introduced as independent variables into a multiple regression analysis. The stepwise model ( R2: 0.113, p = 0.016) retained only A1c levels (β: ‒ 0.333, 95% confidence interval [CI]: −3.10 to −0.33) as significant predictor of the percentage of nighttime decrease in mean BP. Conclusions A non-dipping pattern in BP is very common in patients with type 1 diabetes presenting with subclinical CAN and is associated with a poorer metabolic control. On the contrary, coexistence of arterial stiffness is not associated with abnormalities in circadian BP regulation.

Supplemental Digital Content is available in the text., Background: Hutchinson-Gilford progeria syndrome (HGPS) is a rare disorder characterized by premature aging and death mainly because of myocardial infarction, stroke, or heart failure. The disease is provoked by progerin, a variant of lamin A expressed in most differentiated cells. Patients look healthy at birth, and symptoms typically emerge in the first or second year of life. Assessing the reversibility of progerin-induced damage and the relative contribution of specific cell types is critical to determining the potential benefits of late treatment and to developing new therapies. Methods: We used CRISPR-Cas9 technology to generate LmnaHGPSrev/HGPSrev (HGPSrev) mice engineered to ubiquitously express progerin while lacking lamin A and allowing progerin suppression and lamin A restoration in a time- and cell type–specific manner on Cre recombinase activation. We characterized the phenotype of HGPSrev mice and crossed them with Cre transgenic lines to assess the effects of suppressing progerin and restoring lamin A ubiquitously at different disease stages as well as specifically in vascular smooth muscle cells and cardiomyocytes. Results: Like patients with HGPS, HGPSrev mice appear healthy at birth and progressively develop HGPS symptoms, including failure to thrive, lipodystrophy, vascular smooth muscle cell loss, vascular fibrosis, electrocardiographic anomalies, and precocious death (median lifespan of 15 months versus 26 months in wild-type controls, P

Copeptin is a stable peptide derived from cleavage of the precursor of arginine-vasopressin (AVP), [1,2] that can be easily measured by using a simple rapid assay. Copeptin has been related with an increased incidence of stroke and cardiovascular mortality in individuals with diabetes, mainly type 2 [3,4]. It is also a risk predictor for end-stage renal disease, coronary heart disease, and all-cause mortality in people with type 1 diabetes (T1DM) [5]. However, we have recently reported that copeptin does not appear to be a reliable marker for the screening of asymptomatic peripheral arterial disease in a population of patients with T1DM [6]. We here explore the putative relationships between serum copeptin level and carotid intima-media thickness (cIMT) in asymptomatic T1DM-patients comprehensively investigated for subclinical carotid atherosclerosis.

Aim: To clarify whether autoantibodies against glutamic acid decarboxylase (GAD-ab) are associated with cardiac autonomic neuropathy (CAN). Methods: 319 consecutive patients with type 1 diabetes (DM1) were included. We assessed CAN by responses of heart rate variability to deep breathing (E/I index), Valsalva maneuver, standing (30:15 ratio), low-frequency activity (LFa), and high-frequency activity (HFa). We assayed GAD-ab by immunoprecipitation radioligand. Results: Mean age was of 40 ± 14 years, with 20 ± 12 years as mean duration of diabetes. Mean A1c was 7.2 ± 1.0%. We confirmed CAN in 66 patients resulting in a 21% (95% CI 17-25) prevalence of asymptomatic CAN. GAD-ab were present in 199 individuals (62%), with no significant differences as a function of the presence of CAN or not [44 (70%) vs. 144 (66%), respectively; P = 0.544]. GAD-ab titles were similar between both groups (431 ± 695 U/mL in CAN subjects vs. 390 ± 662 U/mL in subjects without CAN, P = 0.688). Considering all patients as a whole, GAD-ab titles only correlated with duration of DM1 (r = -0.346, P < 0.001). HFa correlated with age (r = -0.523, P < 0.001), duration of DM1 (r = -0.374, P < 0.001), and A1c (r = -0.261, P < 0.001), but not with GAD-ab. Similarly, LFa correlated with age (r = -0.473, P < 0.001), duration of DM1 (r = -0.223, P < 0.001), and A1c (r = -0.294, P < 0.001). Age, sex, A1c, duration of DM1, GAD-ab, and systolic blood pressure were introduced as independent variables into a binary regression analysis. The stepwise model (R2: 0.106, P < 0.001) retained only age (β: -0.933, 95% CI: 0.933-0.976) as the significant predictor of the presence of CAN. Conclusion: GAD-ab unlikely have a role as a CAN marker in patients with DM1. Disclosure L. Nattero-chávez: None. E. Lecumberri: None. H. F. Escobar-morreale: Consultant; Self; InsudPharma. M. Luque-ramírez: None. J. Quiñones silva: None. L. Montanez: None. E. Fernández durán: None. B. Dorado avendaño: None. C. Sánchez rodríguez: None. M. Lorenzo: None. M. Fernández argüeso: None. N. Bengoa rojano: None. Funding Menarini International Foundation

Introduction: Increased prevalence of functional hyperandrogenism/polycystic ovary syndrome (PCOS) has been previously described in type 1 diabetes (T1DM) patients. However, the reported prevalence rates vary depending on different diagnostic criteria. Our study aim was to determine the prevalence of PCOS and hyperandrogenic traits in premeopausal women with T1DM. Methods: A cross-sectional study was conducted at a diabetes outpatient clinic in a tertiary Hospital from Madrid, Spain. We recruited consecutive premenopausal woman with T1DM, and menarche at least 2 years before the study. For this preliminary report, we included 40 patients. History data collection, physical exam, a complete hormonal analysis in follicular phase (including d4-androstenedione, DHEA-S, total testosterone, SHBG, 17-OH-progesterone, FSH, LH, prolactin, and TSH), and ovarian ultrasound were performed. Results: Mean age of patients was of 28 ± 8 yr; mean duration of T1DM was of 13 ± 8 yr-old. Twenty-seven patients were on multiple daily insulin therapy, while 13 of them were on continuous subcutaneous insulin infusion. Mean BMI was of 23.8 ± 3.7 Kg/m2. Mean daily insulin dose was 0.64 ± 0.2 U/kg and mean A1C was 7.5 ± 1.8%. PCOS was diagnosed in 7 patients resulting in a 18% (95% CI: 8; 32) prevalence of PCOS according to NIH-Rotterdam, and in 6 patients resulting in a 15% (95% CI: 7; 29) prevalence of PCOS according to AES-PCOS criteria. Three patients [7% (95% CI: 3; 20)] had isolated polycystic ovarian morphology (PCOM), and 7 patients [18% (95% CI: 8; 32)] presented with oligo-amenorrhea (ovulatory dysfunction-WHO class 2). Conclusion: Our preliminary data show a significant increase in prevalence of PCOS in premenopausal women with T1DM with respect to the general population. Disclosure A. Bayona: None. L. Nattero-chávez: None. E. Fernández durán: None. B. Dorado avendaño: None. H. F. Escobar-morreale: Consultant; Self; InsudPharma. M. Luque-ramírez: None.

Objective: To investigate the effect of 6m CFTR modulators treatment on insulin secretion in CF patients. Methods: 21 CF patients (17 Phe.508del hom) were included in a prospective, observational study. After 6m of lumacaftor/tezacaftor+ivacaftor, AUC of glucose, insulin and C peptide were evaluated by using 5-point OGTT. GLM repeated measures test performed. Results: 21 patients initially participated, 3 were excluded: 1 had no basal OTTG, and 2 had drug intolerance. Median age 14,7yr (ICR 8,5-26,0); 57% ≥13 yr and 57% male. Initially: 8 had normal glucose metabolism (N), 1 indeterminate (IND), 2 abnormal glucose tolerance (AGT), 2 IND+AGT, and 5 CFRD. After 6m: in the N group: 5 (62,5%; 100% Conclusion: Increase in AUC of insulin and C-peptide were observed after 6m with CFTR modulators, suggesting a potential role in improving insulin secretion. AUC of glucose should be analyzed prospectively. Disclosure E. Lecumberri: None. R. Y. Yelmo: None. M. Garriga: None. A. F. Lamas: None. S. Vicente: Consultant; Self; Abbott, Fresenius Medical Care, Mead Johnson Nutrition. M. Martin-frias: None. L. Nattero-chavez: None. V. Martinez-vaello: None. L. Maiz: None. C. Sanchez rodriguez: None. M. Lorenzo: None. B. Dorado avendano: None.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disease that recapitulates many symptoms of physiological aging and precipitates death. Patients develop severe vascular alterations, mainly massive vascular smooth muscle cell loss, vessel stiffening, calcification, fibrosis, and generalized atherosclerosis, as well as electrical, structural, and functional anomalies in the heart. As a result, most HGPS patients die of myocardial infarction, heart failure, or stroke typically during the first or second decade of life. No cure exists for HGPS, and therefore it is of the utmost importance to define the mechanisms that control disease progression in order to develop new treatments to improve the life quality of patients and extend their lifespan. Since the discovery of the HGPS-causing mutation, several animal models have been generated to study multiple aspects of the syndrome and to analyze the contribution of different cell types to the acquisition of the HGPS-associated cardiovascular phenotype. This review discusses current knowledge about cardiovascular features in HGPS patients and animal models and the molecular and cellular mechanisms through which progerin causes cardiovascular disease. This study was supported by grants to V.A. from the Spanish Ministerio de Ciencia e Innovación (MCIN) (PID2019-108489RB-I00) and the Instituto de Salud Carlos III (ISCIII) (grant AC17/00067, as coordinator of the E-Rare Joint Transnational call 2017 project “TREAT-HGPS,” European Union Horizon 2020 Framework Programme) with co-funding from the European Regional Development Fund (ERDF, “A way to build Europe”), the Progeria Research Foundation (Award PRF 2019–77), and the Fundación Marató TV3 (38/C/2020). I.B. was supported by the Comunidad Autónoma de Madrid (grant 2017-T1/BMD-5247). The CNIC is supported by the MCIN, the ISCIII, and the Pro CNIC Foundation. Sí

The male predominance of cardiovascular disease is overridden by the presence of diabetes, as the sex-related burden of cardiovascular disease increases in postmenopausal women with diabetes (1). Sexual dimorphism in cardiovascular autonomic neuropathy (CAN) might contribute to these findings (1). We conducted a cross-sectional study hypothesizing that 1 ) the prevalence of CAN is different among women and men with type 1 diabetes, 2 ) such a difference is dependent on the pre- or postmenopausal status of women, and 3 ) sex steroids show opposite associations with CAN depending on sex. In this study, we recruited 279 consecutive patients with type 1 diabetes attending our clinic (ClinicalTrials.gov identifier NCT02910271) (2,3). Inclusion and exclusion criteria are detailed elsewhere (2,3). Assuming a global prevalence of CAN of ∼30% in our population of patients with type 1 diabetes (2) and setting α at 0.05 and β at 0.20 for a two-sided test, the inclusion of at least 160 men and 120 women was enough to identify a difference in prevalence among them above 15% (www.imim.cat/ofertadeserveis/software-public/granmo/). The study protocol conformed to the ethical guidelines of the Declaration of Helsinki and was approved by the local ethics committee of Hospital Universitario Ramon y Cajal (approval date 22 January 2016, protocol ID 464/15). Informed consent was obtained from all participants included in the study. Patients did not receive any economic compensation for their participation. For …

Objective As copeptin is associated with lower-extremity amputation in patients with type 1 diabetes mellitus (T1DM), our study aimed to address the putative association between copeptin and asymptomatic peripheral artery disease (aPAD) in those patients. Design and methods This observational cross-sectional study included 112 patients with T1DM from a larger cohort (ClinicalTrials.gov: NCT02910271), selected (1:2) as per the presence of aPAD (n = 37) or not (n = 75). aPAD was evaluated by ankle–brachial index (ABI), toe–brachial index (TBI), and peripheral Doppler ultrasound. The two groups of patients were matched by age, gender distribution and duration of T1DM. Fasting serum copeptin was measured by high-sensitivity ELISA, and its relationships with clinical and biochemical variables as well as aPAD were evaluated too. Results The study population was aged 42 ± 8 years, duration of T1DM was 27 ± 7 years, and mean HbA1c was 7.7 ± 1.1%. No significant differences in copeptin concentrations were found between patients with or without aPAD (16.9 ± 10.8 vs 17.3 ± 14.7 pmol/L, respectively; P = 0.462). Considering all patients as a whole, copeptin correlated with systolic blood pressure (SBP; ρ = −0.209, P = 0.027), eGFR ρ = −0.271, P = 0.004), and serum sodium (ρ = −0.208, P = 0.027), but not with ABI (ρ = −0.068, P = 0.476). Stepwise multiple linear regression analysis (R2: 0.059; P = 0.035) retained SBP (β: −0.219, 95% CI: −1.391; −0.089) as the only significant predictor of copeptin concentration. Conclusion As serum copeptin does not appear to be associated with aPAD in patients with T1DM, further studies are now needed to elucidate whether it has any other potential role to play in the subclinical vascular disease of this patient population.

Aim: Describe the relationships between cardiovascular autonomic neuropathy (CAN) and glucose variability (GV) in patients with DM1 on continuous subcutaneous insulin infusion therapy (CSII). Methods Observational retrospective study (n=89). CAN was assessed by heart rate variability (HRV) in response to deep breathing, standing, Valsalva’s maneuver, and the amplitude of the low (LFa) and high (HFa) frequency of HRV. The standard deviation (SD), the coefficient of variation (CV) and time in range (TIR) were calculated from the capillary blood measurements downloaded of CSII (the mean of glucose capillary assessments was 5.8/day). Results The mean age was 38±13 and their average duration of DM1 was 22±10 years. The prevalence of CAN was 36% (95%CI: 27-46). The population characteristics according to the presence of CAN are summarized in Table 1. The age and the duration of diabetes correlated negatively with LFa (r= -0.496, P < 0.001) and HFa (r= -0.510, P < 0.001). Separate multivariate binary logistic models were constructed for clinical and GV parameters introducing CAN as dependent variable. Only the age (expB 1.1; CI 1.04-1.15. P < 0.001) was associated with CAN in the fully adjusted model. Conclusions Age and duration of diabetes, but not GV parameters, appear to be associated with CAN in our cohort of patients with DM1 on CSII. Disclosure L. Nattero-Chávez: None. J. Quiñones Silva: None. N. Bengoa: None. M. Fernández Argüeso: None. L. Montanez: None. E. Fernández Durán: None. B. Dorado: None. B. Arias Zamorano: None. E. Lecumberri: None. M. Luque Ramírez: None. Funding Menarini Group

Cardiovascular disease (CVD) is the main cause of death worldwide, and aging is its leading risk factor. Aging is much accelerated in Hutchinson&ndash, Gilford progeria syndrome (HGPS), an ultra-rare genetic disorder provoked by the ubiquitous expression of a mutant protein called progerin. HGPS patients die in their teens, primarily due to cardiovascular complications. The primary causes of age-associated CVD are endothelial dysfunction and dysregulated vascular tone, however, their contribution to progerin-induced CVD remains poorly characterized. In the present study, we found that progeroid LmnaG609G/G609G mice with ubiquitous progerin expression show both endothelial dysfunction and severe contractile impairment. To assess the relative contribution of specific vascular cell types to these anomalies, we examined LmnaLCS/LCSTie2Cretg/+ and LmnaLCS/LCSSm22&alpha, Cretg/+ mice, which express progerin specifically in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. Whereas vessel contraction was impaired in mice with VSMC-specific progerin expression, we observed no endothelial dysfunction in mice with progerin expression restricted to VSMCs or ECs. Vascular tone regulation in progeroid mice was ameliorated by dietary sodium nitrite supplementation. Our results identify VSMCs as the main cell type causing contractile impairment in a mouse model of HGPS that is ameliorated by nitrite treatment.

Aging is the main risk factor for cardiovascular and metabolic diseases, which have become a global concern as the world population ages. These diseases and the aging process are exacerbated in Hutchinson–Gilford progeria syndrome (HGPS or progeria). Here, we evaluated the cardiometabolic disease in animal models of premature and normal aging with the aim of identifying alterations that are shared or specific to each condition. Despite differences in body composition and metabolic markers, prematurely and normally aging mice developed heart failure and similar cardiac electrical abnormalities. High‐throughput proteomics of the hearts of progeric and normally aged mice revealed altered protein oxidation and glycation, as well as dysregulated pathways regulating energy metabolism, proteostasis, gene expression, and cardiac muscle contraction. These results were corroborated in the hearts of progeric pigs, underscoring the translational potential of our findings, which could help in the design of strategies to prevent or slow age‐related cardiometabolic disease., We have evaluated cardiac and metabolic disease in animal models of aging. Despite differences in body composition, progeric and normally aged mice developed heart failure and similar cardiac electrical alterations. High‐throughput proteomics of the hearts of these animals revealed dysregulation of energy metabolism, proteostasis, gene expression, and cardiac contraction pathways. These results were corroborated in progeric pigs, underscoring the translational potential of our findings.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients. This project was mainly supported by an Established Investigator Award from the Progeria Research Foundation (2014-52), and from the Spanish Ministerio de Ciencia, Innovación y Universidades (MCIU), and the European Regional Development Fund (FEDER, “A way to build Europe”) (SAF2016-79490-R, CB16/11/00405). Ana Barettino has a predoctoral contract from MCIU (BES-2017-079705). Work at Universidad de Murcia is supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia (20040/GERM/16). The CNIC is supported by the MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505). Sí

This study was supported by Instituto de Salud Carlos III (Grant RD12/0042/0028 to Dr. Andres), with cofunding from Fondo Europeo de Desarrollo Regional (FEDER), and by a noncompetitive, unrestricted grant shared equally between the CNIC and Banco de Santander. The PESA study is a noncommercial study that is independent of the health care and pharmaceutical industry. The Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro CNIC Foundation support the CNIC. The MINECO RETOS Program and the Fundacion Botin fund work in Dr. Blasco0s group. The CNIC and CNIO are recipients of Centro de Excelencia Severo Ochoa awards from MINECO (SEV-2015-0505 and SEV-2015-0510, respectively). The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Peter M. Nilsson, MD, served as Guest Editor for this paper.

Lamin A is a nuclear intermediate filament protein with important structural and regulatory roles in most differentiated mammalian cells. Excessive accumulation of its precursor prelamin A or the mutant form called 'progerin' causes premature aging syndromes. Progeroid 'laminopathies' are characterized by severe cardiovascular problems (cardiac electrical defects, vascular calcification and stiffening, atherosclerosis, myocardial infarction, and stroke) and premature death. Here, we review studies in cell and mouse models and patients that are unraveling how abnormal prelamin A and progerin accumulation accelerates cardiovascular disease and aging. This knowledge is essential for developing effective therapies to treat progeria and may help identify new mechanisms underlying normal aging. Work in the laboratory of V.A. is supported by the Spanish Ministerio de Economia, Industria y Competitividad (MINECO) (SAF2013-46663-R) and the Instituto de Salud Carlos III (RD12/0042/0028) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER), the Fundacio Marato TV3 (122/C/2015), and the Progeria Research Foundation (Established Investigator Award 2014-52). The CNIC is supported by the MINECO and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). Sí

Leucocyte telomere length (LTL) shortening is associated with cardiovascular ischemic events and mortality in humans, but data on its association with subclinical atherosclerosis are scarce. Whether the incidence and severity of subclinical atherosclerosis are associated with the abundance of critically short telomeres, a major trigger of cellular senescence, remains unknown.The authors conducted a cross-sectional exploration of the association between subclinical atherosclerosis burden and both average LTL and the abundance of short telomeres (%LTL3 kb).Telomere length was assessed by high-throughput quantitative fluorescence in situ hybridization in circulating leukocytes from 1,459 volunteers without established cardiovascular disease (58% men, 40 to 54 years of age) from the PESA (Progression of Early Subclinical Atherosclerosis) study. Subclinical atherosclerosis was evaluated by coronary artery calcium scan and 2-dimensional/3-dimensional ultrasound in different aortic territories. Statistical significance of differences among multiple covariates was assessed with linear regression models. Independent associations of telomere parameters with plaque presence were evaluated using general linear models.In men and women, age was inversely associated with LTL (Pearson's r = -0.127, p 0.001) and directly with %LTL3 kb (Pearson's r = 0.085; p = 0.001). Short LTL reached statistical significance as a determinant of total and femoral plaque in men, but not in women. However, this association was not sustained after adjustment for age or additional adjustment for cardiovascular risk factors. No significant independent association was found between %LTL3 kb and plaque burden. Serum-oxidized low-density lipoprotein levels were directly associated with %LTL3 kb in men (p = 0.008) and women (p 0.001).In a cross-sectional study of a middle-aged population, average LTL and short telomere load are not significant independent determinants of subclinical atherosclerosis. Longitudinal follow-up of PESA participants will assess long-term associations between telomere length and progression of subclinical atherosclerosis.

Deficiency of thymidine kinase 2 (TK2) is a frequent cause of isolated myopathy or encephalomyopathy in children with mitochondrial DNA (mtDNA) depletion. To determine the bases of disease onset, organ specificity and severity of TK2 deficiency, we have carefully characterized Tk2 H126N knockin mice (Tk2−/−). Although normal until postnatal day 8, Tk2−/− mice rapidly develop fatal encephalomyopathy between postnatal days 10 and 13. We have observed that wild-type Tk2 activity is constant in the second week of life, while Tk1 activity decreases significantly between postnatal days 8 and 13. The down-regulation of Tk1 activity unmasks Tk2 deficiency in Tk2−/− mice and correlates with the onset of mtDNA depletion in the brain and the heart. Resistance to pathology in Tk2 mutant organs depends on compensatory mechanisms to the reduced mtDNA level. Our analyses at postnatal day 13 have revealed that Tk2−/− heart significantly increases mitochondrial transcript levels relative to the mtDNA content. This transcriptional compensation allows the heart to maintain normal levels of mtDNA-encoded proteins. The up-regulation in mitochondrial transcripts is not due to increased expression of the master mitochondrial biogenesis regulators peroxisome proliferator-activated receptor-gamma coactivator 1 alpha and nuclear respiratory factors 1 and 2, or to enhanced expression of the mitochondrial transcription factors A, B1 or B2. Instead, Tk2−/− heart compensates for mtDNA depletion by down-regulating the expression of the mitochondrial transcriptional terminator transcription factor 3 (MTERF3). Understanding the molecular mechanisms that allow Tk2 mutant organs to be spared may help design therapies for Tk2 deficiency.

Replication and repair of DNA require equilibrated pools of deoxynucleoside triphosphate precursors. This concept has been proven by in vitro studies over many years, but in vivo models are required to demonstrate its relevance to multicellular organisms and to human diseases. Accordingly, we have generated thymidine phosphorylase (TP) and uridine phosphorylase (UP) double knockout (TP(-/-)UP(-/-)) mice, which show severe TP deficiency, increased thymidine and deoxyuridine in tissues and elevated mitochondrial deoxythymidine triphosphate. As consequences of the nucleotide pool imbalances, brains of mutant mice developed partial depletion of mtDNA, deficiencies of respiratory chain complexes and encephalopathy. These findings largely account for the pathogenesis of mitochondrial neurogastrointestinal encephalopathy (MNGIE), the first inherited human disorder of nucleoside metabolism associated with somatic DNA instability.

Methods for staining tissues with Oil Red O and hematoxylin-eosin are classical histological techniques that are widely used to quantify atherosclerotic burden in mouse tissues because of their ease of use, reliability, and the large amount of information they provide. These stains can provide quantitative data about the impact of a genetic or environmental factor on atherosclerotic burden and on the initiation, progression, or regression of the disease, and can also be used to evaluate the efficacy of drugs designed to prevent or treat atherosclerosis. This chapter provides protocols for quantifying atherosclerotic burden in mouse aorta and aortic root, including methods for dissection, Oil Red O staining, hematoxylin-eosin staining, and image analysis.

A label-free compact method for performing photonic characterization of "healthy" versus "diseased" arteries has been developed. It permits the detection of atherosclerotic lesion in living mouse arteries. Using this prototype, we observed that the spectral response (photonic fingerprint, PIN) obtained from aortas of wild-type mice differs from the response of ApoE-KO mice fed with high-fat diet (an atheroprone mouse model). Benchmark of the results against gold standard was performed by staining the aortas with Oil-Red-O to visualize atherosclerotic plaques.

The LiPhos project (EU FP7 Grant Agreement No.: 317916, www.liphos.eu) aims to develop three different biophotonic diagnosis tools (BDTs), based on living photonics, namely: single layer living photonics (SLLP), single cell analysis (SCA); and multi-layer living photonics (MLLP). By Measuring of what we term the Photonic Fingerprint (or PIN), of the cells in such BDTs, should make it is possible to differentiate between healthy and non-healthy cell or tissue states. Moreover, the effect of specific drugs and pro-inflammatory agents could be assessed. This concept is currently being applied to the diagnosis of cardiovascular diseases (CVD).

Methods for staining tissues with Oil Red O and hematoxylin-eosin are classical histological techniques that are widely used to quantify atherosclerotic burden in mouse tissues because of their ease of use, reliability, and the large amount of information they provide. These stains can provide quantitative data about the impact of a genetic or environmental factor on atherosclerotic burden and on the initiation, progression, or regression of the disease, and can also be used to evaluate the efficacy of drugs designed to prevent or treat atherosclerosis. This chapter provides protocols for quantifying atherosclerotic burden in mouse aorta and aortic root, including methods for dissection, Oil Red O staining, hematoxylin-eosin staining, and image analysis. The authors thank Simon Bartlett for editorial assistance. Work in VA’s laboratory is supported by grants SAF2013-46663-R and RD12/0042/0028 from the Spanish Ministerio de Economía y Competitividad (MINECO) with co-funding from the Fondo Europeo de Desarrollo Regional (FEDER), the European Commission (Liphos, Grant Agreement No. 317916), and the Progeria Research Foundation (Established Investigator Award). The CNIC is supported by the MINECO and the Pro-CNIC Foundation.

Current evidence suggests that multiple sclerosis (MS) results from an autoimmune response mediated by T lymphocytes, which would be activated in the peripheral blood and migrate into the central nervous system. NFkappaB and AP-1 are two main transcription factors involved in T-cell activation. To investigate possible alterations in the activity of these factors in MS individuals, we have assayed NFkappaB and AP-1 DNA binding activity in peripheral blood mononuclear cells (PBMC). Binding activity was analyzed by gel mobility shift assay in MS patients compared with controls. No significant differences were found between the two groups, indicating no evidence of abnormalities associated with MS in NFkappaB or AP-1 binding activities in PBMC, both basally and after PMA+anti-CD3 antibody induction.

Primary human CoQ10 deficiencies are clinically heterogeneous diseases caused by mutations in PDSS2 and other genes required for CoQ10 biosynthesis. Our in vitro studies of PDSS2 mutant fibroblasts, with

Because deoxyribonucleoside triphosphates (dNTPs) are the critical substrates for DNA replication and repair, dNTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a maternally inherited mitochondrial syndrome characterized by seizures, migrainous headaches, lactic acidosis, vomiting, and recurrent stroke-like episodes. Patients often suffer from cognitive dysfunction of unclear pathogenesis. In this study, we explored a possible link between cognitive dysfunction and hippocampal expression of calbindin D(28KD) (CB), a high affinity calcium-binding protein, in four MELAS patients, using post mortem hippocampal tissues. We found significantly reduced CB levels in all patients by immunohistochemistry, Western blot, and quantitative real-time PCR. Reduction in CB expression has been associated with aging and with neurodegenerative disorders, including Alzheimer's disease. We postulate that the reduced CB expression may play a role in the cognitive abnormalities associated with MELAS.

The mitochondrial DNA (mtDNA) depletion syndrome comprises a clinically heterogeneous group of diseases characterized by reductions of the mtDNA abundance, without associated point mutations or rearrangements. We have developed the first in vitro model to study of mtDNA depletion due to reduced mitochondrial thymidine kinase 2 gene ( TK2 ) expression in order to understand the molecular mechanisms involved in mtDNA depletion syndrome due to TK2 mutations. Small interfering RNA targeting TK2 mRNA was used to decrease TK2 expression in Ost TK1 − cells, a cell line devoid of endogenous thymidine kinase 1 (TK1). Stable TK2-deficient cell lines showed a reduction of TK2 levels close to 80%. In quiescent conditions, TK2-deficient cells showed severe mtDNA depletion, also close to 80% the control levels. However, TK2-deficient clones showed increased cytochrome c oxidase activity, higher cytochrome c oxidase subunit I transcript levels and higher subunit II protein expression respect to control cells. No alterations of the deoxynucleotide pools were found, whereas a reduction in the expression of genes involved in nucleoside/nucleotide homeostasis (human equilibrative nucleoside transporter 1, thymidine phosphorylase) and mtDNA maintenance (DNA-polymerase γ, mitochondrial transcription factor A) was observed. Our findings highlight the importance of cellular compensatory mechanisms that enhance the expression of respiratory components to ensure respiratory activity despite profound depletion in mtDNA levels.