Rationale. Liver dysfunction is the major determinant of survival in patients with Acute Respiratory Distress Syndrome (ARDS). The liver-lung axis plays a role in the development of lung injury. This study aimed at determining the gene expression profile in the lung of rats with cirrhosis at different disease stages. Material and method. Cirrhosis was induced in adult rats by oral gavage with carbon tetrachloride (CCl4) twice a week for 8 or 12 weeks. Rats gavaged with water were used as controls (n= 4). After euthanasia, the liver weight/body weight ratio was measured to identify liver atrophy. Depending on the presence of liver atrophy, the rats were then classified into CCL4-No atrophy (n= 4) and CCL4-Atrophy (n= 4). Gene expression in the lung was assessed by RNAseq. Differential expression analysis of the mRNA sequencing data was conducted using EdgeR. Pathway enrichment analysis was performed using web-based tools (Reactome). Differentially expressed genes (DEGs) were defined as those with adjusted p value < 0.05. Results. All rats treated with CCL4 had severe fibrosis, cirrhosis of the liver and portal hypertension. Compared with controls, there were 40 DEGs in the lungs of CCL4-No atrophy rats and up to 997 DEGs in the lungs of rats showing liver atrophy, with only 18 DEGs shared between both groups. Interestingly, Reactome analysis of DEGs specific to each group identified inflammatory-related pathways as the most significantly deregulated in the lungs of CCL4-Atrophy rats, whereas the most deregulated pathways in CCL4-No atrophy rats were metabolism-related. Reactome analysis of the 18 DEGs shared between both groups were also metabolism-related. Compared with the control group, most of the DEGs in both CCL4 groups were upregulated (n= 22 in CCL4-No atrophy, n= 757 in CCL4-atrophy, with 12 genes in common). The number of downregulated DEGs in CCL4-No atrophy rats were 17, whereas the number of downregulated DEGs in rats with liver atrophy were much lower (n= 241) than those upregulated, with only 5 downregulated DEGs shared between both CCL4 groups. Conclusions. Development of liver atrophy in rats with CCL4-induced cirrhosis significantly impacted the gene expression profile in the lung. The results also suggest that the development of liver atrophy in cirrhotic rats is associated with a shift in the type of signaling pathways that are deregulated in the lung from metabolism-related to inflammation-related pathways.

Hutchinson-Gilford progeria syndrome (HGPS) is an ultrarare genetic disease caused by progerin, a broadly expressed mutant variant of lamin A protein that accelerates aging and leads to premature death typically in adolescence. Progerin affects many organs and reproduces many characteristics of physiological aging, with the main cause of death in HGPS being atherosclerotic cardiovascular disease (CVD). Due to the rarity of HGPS, advances in understanding the disease and progress toward new therapeutic approaches are crucially dependent on preclinical models. We discuss recent research developments from a variety of HGPS experimental systems, with a special focus on in vivo studies of the role of vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) that are key players in atherosclerosis.

Outer retinal function depends on two supporting tissues: the retinal pigment epithelium (RPE) and the choroid. Limited molecular information is available on the intercellular networks that sustain RPE/choroid tissue in both healthy and pathological states. Galectin-1 (Gal1), a β-galactoside-binding lectin, has recently emerged as a key regulator of angiogenesis and a potential therapeutic target in vascular pathologies, including age-related macular degeneration. Here, we studied the expression of Gal1 in the outer retina and its regulatory role in the RPE/choroid under physiological and pathological conditions. Our findings indicate that Gal1 is predominantly associated with stromal cells in the RPE/choroid. In Gal1-deficient (Lgals1-/-) mice, the RPE/choroid ultrastructure and gene expression profiles were altered, and choroidal explants exhibited reduced sprouting compared to those of wild-type mice. Consistently, recombinant Gal1 promoted choroidal sprouting under hypoxic conditions, and stromal-like cells modulated pro-angiogenic and antiangiogenic gene expression in vitro under pathological conditions. Interestingly, Gal1 was also expressed by the RPE, with apical secretion under normoxia that shifted toward a basolateral phenotype under hypoxia. These findings identify stromal-like cells and RPE as key sources of Gal1 in the choroid, highlighting its distinct roles in maintaining RPE/choroid homeostasis in healthy or pathological microenvironments.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here, we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder caused by a mutation in the LMNA gene that provokes the synthesis of progerin, a mutant version of the nuclear protein lamin A that accelerates aging and precipitates death. The most clinically relevant feature of HGPS is the development of cardiac anomalies and severe vascular alterations, including massive loss of vascular smooth muscle cells, increased fibrosis, and generalized atherosclerosis. However, it is unclear if progerin expression in endothelial cells (ECs) causes the cardiovascular manifestations of HGPS. To tackle this question, we generated atherosclerosis-free mice (LmnaLCS/LCSCdh5-CreERT2) and atheroprone mice (Apoe-/-LmnaLCS/LCSCdh5-CreERT2) with EC-specific progerin expression. Like progerin-free controls, LmnaLCS/LCSCdh5-CreERT2 mice did not develop heart fibrosis or cardiac electrical and functional alterations, and had normal vascular structure, body weight, and lifespan. Similarly, atheroprone Apoe-/-LmnaLCS/LCSCdh5-CreERT2 mice showed no alteration in body weight or lifespan versus Apoe-/-LmnaLCS/LCS controls and did not develop vascular alterations or aggravated atherosclerosis. Our results indicate that progerin expression in ECs is not sufficient to cause the cardiovascular phenotype and premature death associated with progeria.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare disease caused by the expression of progerin, an aberrant protein produced by a point mutation in the LMNA gene. HGPS patients show accelerated aging and die prematurely mainly from complications of atherosclerosis such as myocardial infarction, heart failure, or stroke. However, the mechanisms underlying HGPS vascular pathology remain ill-defined. We used single-cell RNA sequencing to characterize the aorta in progerin-expressing Lmna G609G/G609G mice and wild-type controls, with a special focus on endothelial cells (ECs). HGPS ECs showed gene expression changes associated with extracellular matrix alterations, increased leukocyte extravasation, and activation of the yes-associated protein 1/transcriptional activator with PDZ-binding domain (YAP/TAZ) mechanosensing pathway, all validated by different techniques. Atomic force microscopy experiments demonstrated stiffer subendothelial extracellular matrix in progeroid aortae, and ultrasound assessment of live HGPS mice revealed disturbed aortic blood flow, both key inducers of the YAP/TAZ pathway in ECs. YAP/TAZ inhibition with verteporfin reduced leukocyte accumulation in the aortic intimal layer and decreased atherosclerosis burden in progeroid mice. Our findings identify endothelial YAP/TAZ signaling as a key mechanism of HGPS vascular disease and open a new avenue for the development of YAP/TAZ-targeting drugs to ameliorate progerin-induced atherosclerosis.

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 Lmna G609G/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 K V 7 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 K V 7 channels as a candidate target for the treatment of HGPS.

Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare genetic disease characterized by premature aging and death at an average age of 14.6 years. Most HGPS patients are heterozygous carriers of a de novo synonymous mutation in the LMNA gene, which encodes the nuclear proteins lamin A and C. This mutation activates the use of a cryptic splice donor site in exon 11, generating an aberrant lamin A variant called progerin that remains permanently farnesylated and methylated and causes multiple cellular and organismal alterations. HGPS patients appear normal at birth, and the first disease symptoms are growth failure and alopecia, typically appearing in the first or second year of life. Over time, additional symptoms develop and worsen. Patients typical.

Confocal imaging of the mouse aorta is a powerful, indispensable technique for the study of cardiovascular pathology ex vivo. Whole mount en face preparations allow visualization of wide areas of the luminal vessel surface, thus enabling a thorough analysis of multiple cellular and structural features of the endothelial cell-rich intimal layer. This method is a suitable tool for the study of endothelial cell dysfunction and leukocyte infiltration, both of which contribute to the onset of pathological vascular conditions such as atherosclerosis. This chapter provides a complete guide on how to perfuse-fix mouse aorta, dissect the vessel, immunostain target proteins, and carry out en face confocal image acquisition and analysis.

Alpha-2-macroglobulin (A2M) is a protease inhibitor that regulates extracellular matrix (ECM) stability and turnover. Here, we show that A2M is expressed by endothelial cells (ECs) from human eye choroid. We demonstrate that retinal pigment epithelium (RPE)-conditioned medium induces A2M expression specifically in ECs. Experiments using chemical inhibitors, blocking antibodies, and recombinant proteins revealed a key role of VEGF-A in RPE-mediated A2M induction in ECs. Furthermore, incubation of ECs with RPE-conditioned medium reduces matrix metalloproteinase-2 gelatinase activity of culture supernatants, which is partially restored after A2M knockdown in ECs. We propose that dysfunctional RPE or choroidal blood vessels, as observed in retinal diseases such as age-related macular degeneration, may disrupt the crosstalk mechanism we describe here leading to alterations in the homeostasis of choroidal ECM, Bruch’s membrane and visual function.

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 Lmna(HGPSrev/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<0.0001). Ubiquitous progerin suppression and lamin A restoration significantly extended lifespan when induced in 6-month-old mildly symptomatic mice and even in severely ill animals aged 13 months, although the benefit was much more pronounced on early intervention (84.5% lifespan extension in mildly symptomatic mice, P<0.0001, and 6.7% in severely ill mice, P<0.01). It is remarkable that major vascular alterations were prevented and lifespan normalized in HGPSrev mice when progerin suppression and lamin A restoration were restricted to vascular smooth muscle cells and cardiomyocytes. Conclusions:HGPSrev mice constitute a new experimental model for advancing knowledge of HGPS. Our findings suggest that it is never too late to treat HGPS, although benefit is much more pronounced when progerin is targeted in mice with mild symptoms. Despite the broad expression pattern of progerin and its deleterious effects in many organs, restricting its suppression to vascular smooth muscle cells and cardiomyocytes is sufficient to prevent vascular disease and normalize lifespan.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.