Supplementation with a New Standardized Extract of Green and Black Tea Exerts Antiadipogenic Effects and Prevents Insulin Resistance in Mice with Metabolic Syndrome.

Insulin resistance is one of the main characteristics of metabolic syndrome (MetS) and the main cause of the development of type II diabetes. The high prevalence of this syndrome in recent decades has made it necessary to search for preventive and therapeutic agents, ideally of natural origin, with fewer side effects than conventional pharmacological treatments. Tea is widely known for its medicinal properties, including beneficial effects on weight management and insulin resistance. The aim of this study was to analyze whether a standardized extract of green and black tea (ADM® Complex Tea Extract (CTE)) prevents the development of insulin resistance in mice with MetS. For this purpose, C57BL6/J mice were fed for 20 weeks with a standard diet (Chow), a diet with 56% kcal from fat and sugar (HFHS) or an HFHS diet supplemented with 1.6% CTE. CTE supplementation reduced body weight gain, adiposity and circulating leptin levels. Likewise, CTE also exerted lipolytic and antiadipogenic effects in 3T3-L1 adipocyte cultures and in the C. elegans model. Regarding insulin resistance, CTE supplementation significantly increased plasma adiponectin concentrations and reduced the circulating levels of insulin and the HOMA-IR. Incubation of liver, gastrocnemius muscle and retroperitoneal adipose tissue explants with insulin increased the pAkt/Akt ratio in mice fed with Chow and HFHS + CTE but not in those fed only with HFHS. The greater activation of the PI3K/Akt pathway in response to insulin in mice supplemented with CTE was associated with a decrease in the expression of the proinflammatory markers Mcp-1, IL-6, IL-1ß or Tnf-α and with an overexpression of the antioxidant enzymes Sod-1, Gpx-3, Ho-1 and Gsr in these tissues. Moreover, in skeletal muscle, mice treated with CTE showed increased mRNA levels of the aryl hydrocarbon receptor (Ahr), Arnt and Nrf2, suggesting that the CTE's insulin-sensitizing effects could be the result of the activation of this pathway. In conclusion, supplementation with the standardized extract of green and black tea CTE reduces body weight gain, exerts lipolytic and antiadipogenic effects and reduces insulin resistance in mice with MetS through its anti-inflammatory and antioxidant effects.

In vivo grading of lipids in fatty liver by near-infrared autofluorescence and reflectance.

The prevalence of nonalcoholic fatty liver (NAFLD) is rapidly increasing worldwide. When untreated, it may lead to complications such as liver cirrhosis or hepatocarcinoma. The diagnosis of NAFLD is usually obtained by ultrasonography, a technique that can underestimate its prevalence. For this reason, physicians aspire for an accurate, cost-effective, and noninvasive method to determine both the presence and the specific stage of the NAFLD. In this paper, we report an integrated approach for the quantitative estimation of the density of triglycerides in the liver based on the use of autofluorescence and reflectance signals generated by the abdomen of obese C57BL6/J mice. Singular value decomposition is applied to the generated spectra and its corresponding regression model provided a determination coefficient of 0.99 and a root mean square error of 240 mg/dl. This, in turn, enabled the quantitative imaging of triglycerides density in the livers of mice under in vivo conditions.

Carob Extract Supplementation Together with Caloric Restriction and Aerobic Training Accelerates the Recovery of Cardiometabolic Health in Mice with Metabolic Syndrome.

Carob, the fruit of Ceratonia siliqua L. exerts antidiabetic, anti-inflammatory, and antioxidant effects and could be a useful strategy for the treatment and/or prevention of metabolic syndrome (MetS). The aim of this study was to analyze whether supplementation with a carob fruit extract (CSAT+®), alone or in combination with aerobic training, accelerates the recovery of cardiometabolic health in mice with MetS subjected to a caloric restriction. For this purpose, mice were fed with a high fat (58% kcal from fat)/high sugar diet for 23 weeks to induce MetS. During the next two weeks, mice with MetS were switched to a diet with a lower caloric content (25% kcal from fat) supplemented or not with CSAT+® (4.8%) and/or subjected to aerobic training. Both caloric reduction and aerobic training improved the lipid profile and attenuated MetS-induced insulin resistance measured as HOMA-IR. However, only supplementation with CSAT+® enhanced body weight loss, increased the circulating levels of adiponectin, and lowered the plasma levels of IL-6. Moreover, CSAT+® supplementation was the only effective strategy to reduce the weight of epidydimal adipose tissue and to improve insulin sensitivity in the liver and in skeletal muscle. Although all interventions improved endothelial function in aorta segments, only supplementation with CSAT+® reduced obesity-induced hypertension, prevented endothelial dysfunction in mesenteric arteries, and decreased the vascular response of aorta segments to the vasoconstrictor AngII. The beneficial cardiometabolic effects of CSAT+® supplementation, alone or in combination with aerobic training, were associated with decreased mRNA levels of pro-inflammatory markers such as MCP-1, TNFα, IL-1ß, and IL-6 and with increased gene expression of antioxidant enzymes, such as GSR, GPX-3, and SOD-1 in the liver, gastrocnemius, retroperitoneal adipose tissue, and aorta. In conclusion, supplementation with CSAT+®, alone or in combination with aerobic training, to mice with MetS subjected to caloric restriction for two weeks enhances body weight loss, improves the lipid profile and insulin sensitivity, and exerts antihypertensive effects through its anti-inflammatory and antioxidant properties.

Supplementation with Two New Standardized Tea Extracts Prevents the Development of Hypertension in Mice with Metabolic Syndrome.

Hypertension is considered to be both a cardiovascular disease and a risk factor for other cardiovascular diseases, such as coronary ischemia or stroke. In many cases, hypertension occurs in the context of metabolic syndrome (MetS), a condition in which other circumstances such as abdominal obesity, dyslipidemia, and insulin resistance are also present. The high incidence of MetS makes necessary the search for new strategies, ideally of natural origin and with fewer side effects than conventional pharmacological treatments. Among them, the tea plant is a good candidate, as it contains several bioactive compounds such as caffeine, volatile terpenes, organic acids, and polyphenols with positive biological effects. The aim of this study was to assess whether two new standardized tea extracts, one of white tea (WTE) and the other of black and green tea (CTE), exert beneficial effects on the cardiovascular alterations associated with MetS. For this purpose, male C57/BL6J mice were fed a standard diet (Controls), a diet high in fats and sugars (HFHS), HFHS supplemented with 1.6% WTE, or HFHS supplemented with 1.6% CTE for 20 weeks. The chromatography results showed that CTE is more concentrated on gallic acid, xanthines and flavan-3-ols than WTE. In vivo, supplementation with WTE and CTE prevented the development of MetS-associated hypertension through improved endothelial function. This improvement was associated with a lower expression of proinflammatory and prooxidant markers, and-in the case of CTE supplementation-also with a higher expression of antioxidant enzymes in arterial tissue. In conclusion, supplementation with WTE and CTE prevents the development of hypertension in obese mice; as such, they could be an interesting strategy to prevent the cardiovascular disorders associated with MetS.

A Nutraceutical Product Based on a Mixture of Algae and Extra Virgin Olive Oils and Olive Leaf Extract Attenuates Sepsis-Induced Cardiovascular and Muscle Alterations in Rats.

Nutraceuticals are products of natural origin widely used for the treatment and/or prevention of some chronic diseases that are highly prevalent in Western countries, such as obesity or type II diabetes, among others. However, its possible use in the prevention of acute diseases that can put life at risk has been poorly studied. Sepsis is an acute condition that causes cardiovascular and skeletal muscle damage due to a systemic inflammatory state. The aim of this work was to evaluate the possible beneficial effect of a new nutraceutical based on a mixture of algae oil (AO) and extra virgin olive oil (EVOO) supplemented with an olive leaf extract (OLE) in the prevention of cardiovascular alterations and skeletal muscle disorders induced by sepsis in rats. For this purpose, male Wistar rats were treated with the nutraceutical or with water p.o. for 3 weeks and after the treatment they were injected with 1mg/kg LPS twice (12 and 4 h before sacrifice). Pretreatment with the nutraceutical prevented the LPS-induced decrease in cardiac contractility before and after the hearts were subjected to ischemia-reperfusion. At the vascular level, supplementation with the nutraceutical did not prevent hypotension in septic animals, but it attenuated endothelial dysfunction and the increased response of aortic rings to the vasoconstrictors norepinephrine and angiotensin-II induced by LPS. The beneficial effects on cardiovascular function were associated with an increased expression of the antioxidant enzymes SOD-1 and GSR in cardiac tissue and SOD-1 and Alox-5 in arterial tissue. In skeletal muscle, nutraceutical pretreatment prevented LPS-induced muscle proteolysis and autophagy and significantly increased protein synthesis as demonstrated by decreased expression of MURF-1, atrogin-1, LC3b and increased MCH-I and MCH -IIa in gastrocnemius muscle. These effects were associated with a decrease in the expression of TNFα, HDAC4 and myogenin. In conclusion, treatment with a new nutraceutical based on a mixture of AO and EVOO supplemented with OLE is useful to prevent cardiovascular and muscular changes induced by sepsis in rats. Thus, supplementation with this nutraceutical may constitute an interesting strategy to reduce the severity and mortality risk in septic patients.

Reliable and Remote Monitoring of Absolute Temperature during Liver Inflammation via Luminescence-Lifetime-Based Nanothermometry.

Temperature of tissues and organs is one of the first parameters affected by physiological and pathological processes, such as metabolic activity, acute trauma, or infection-induced inflammation. Therefore, the onset and development of these processes can be detected by monitoring deviations from basal temperature. To accomplish this, minimally invasive, reliable, and accurate measurement of the absolute temperature of internal organs is required. Luminescence nanothermometry is the ideal technology for meeting these requirements. Although this technique has lately undergone remarkable developments, its reliability is being questioned due to spectral distortions caused by biological tissues. In this work, how the use of bright Ag2 S nanoparticles featuring temperature-dependent fluorescence lifetime enables reliable and accurate measurement of the absolute temperature of the liver in mice subjected to lipopolysaccharide-induced inflammation is demonstrated. Beyond the remarkable thermal sensitivity (≈ 3% °C-1 around 37 °C) and thermal resolution obtained (smaller than 0.3 °C), the results included in this work set a blueprint for the development of new diagnostic procedures based on the use of intracorporeal temperature as a physiological indicator.

In Vivo Near-Infrared Imaging Using Ternary Selenide Semiconductor Nanoparticles with an Uncommon Crystal Structure.

The implementation of in vivo fluorescence imaging as a reliable diagnostic imaging modality at the clinical level is still far from reality. Plenty of work remains ahead to provide medical practitioners with solid proof of the potential advantages of this imaging technique. To do so, one of the key objectives is to better the optical performance of dedicated contrast agents, thus improving the resolution and penetration depth achievable. This direction is followed here and the use of a novel AgInSe2 nanoparticle-based contrast agent (nanocapsule) is reported for fluorescence imaging. The use of an Ag2 Se seeds-mediated synthesis method allows stabilizing an uncommon orthorhombic crystal structure, which endows the material with emission in the second biological window (1000-1400 nm), where deeper penetration in tissues is achieved. The nanocapsules, obtained via phospholipid-assisted encapsulation of the AgInSe2 nanoparticles, comply with the mandatory requisites for an imaging contrast agent-colloidal stability and negligible toxicity-and show superior brightness compared with widely used Ag2 S nanoparticles. Imaging experiments point to the great potential of the novel AgInSe2 -based nanocapsules for high-resolution, whole-body in vivo imaging. Their extended permanence time within blood vessels make them especially suitable for prolonged imaging of the cardiovascular system.

Addition of Olive Leaf Extract to a Mixture of Algae and Extra Virgin Olive Oils Decreases Fatty Acid Oxidation and Synergically Attenuates Age-Induced Hypertension, Sarcopenia and Insulin Resistance in Rats.

Olive-derived products, such as virgin olive oil (EVOO) and/or olive leaf extracts (OLE), exert anti-inflammatory, insulin-sensitizing and antihypertensive properties and may be useful for stabilizing omega 3 fatty acids (n-3 PUFA) due to their high content in antioxidant compounds. In this study, the addition of OLE 4:0.15 (w/w) to a mixture of algae oil (AO) rich in n-3 PUFA and EVOO (25:75, w/w) prevents peroxides formation after 12 months of storage at 30 °C. Furthermore, the treatment with the oil mixture (2.5 mL/Kg) and OLE (100 mg/Kg) to 24 month old Wistar rats for 21 days improved the lipid profile, increased the HOMA-IR and decreased the serum levels of miRNAs 21 and 146a. Treatment with this new nutraceutical also prevented age-induced insulin resistance in the liver, gastrocnemius and visceral adipose tissue by decreasing the mRNA levels of inflammatory and oxidative stress markers. Oil mixture + OLE also attenuated the age-induced alterations in vascular function and prevented muscle loss by decreasing the expression of sarcopenia-related markers. In conclusion, treatment with a new nutraceutical based on a mixture of EVOO, AO and OLE is a useful strategy for improving the stability of n-3 PUFA in the final product and to attenuate the cardiometabolic and muscular disorders associated with aging.

Olive Leaf Extract Supplementation to Old Wistar Rats Attenuates Aging-Induced Sarcopenia and Increases Insulin Sensitivity in Adipose Tissue and Skeletal Muscle.

Aging is associated with increased visceral adiposity and a decrease in the amount of brown adipose tissue and muscle mass, known as sarcopenia, which results in the development of metabolic alterations such as insulin resistance. In this study, we aimed to analyze whether 3-week supplementation with a phenolic-rich olive leaf extract (OLE) to 24 months-old male Wistar rats orally (100 mg/kg) attenuated the aging-induced alterations in body composition and insulin resistance. OLE treatment increased brown adipose tissue and attenuated the aging-induced decrease in protein content and gastrocnemius weight. Treatment with OLE prevented the aging-induced increase in the expression of PPAR-γ in visceral and brown adipose tissues, while it significantly increased the expression of PPAR-α in the gastrocnemius of old rats and reduced various markers related to sarcopenia such as myostatin, HDAC-4, myogenin and MyoD. OLE supplementation increased insulin sensitivity in explants of gastrocnemius and epididymal visceral adipose tissue from aged rats through a greater activation of the PI3K/Akt pathway, probably through the attenuation of inflammation in both tissues. In conclusion, supplementation with OLE prevents the loss of muscle mass associated with aging and exerts anti-inflammatory and insulin-sensitizing effects on adipose tissue and skeletal muscle.

Olive leaf extract supplementation improves the vascular and metabolic alterations associated with aging in Wistar rats.

Olive leaves are rich in bioactive substances which exert anti-inflammatory, antioxidant, insulin-sensitizing and antihypertensive effects. The aim of this study was to analyze the possible beneficial effects of an olive leaf extract (OLE) rich in secoiridoids and phenolic compounds on the aging-induced metabolic and vascular alterations. Three experimental groups of rats were used: 3-month-old rats, 24-month-old rats and 24-month-old rats supplemented 21 days with OLE (100 mg/kg). Administration of OLE to aged rats decreased the weight of adrenal glands and prevented the aging-induced loss of body weight and muscle mass. In the serum, OLE reduced the circulating levels of LDL-cholesterol and IL-6 and increased the concentrations of leptin and adiponectin. In the liver OLE attenuated the decreased gene expression of SOD-1, GSR, GCK and GSK-3ß and reduced the aging-induced overexpression of NOX-4, Alox-5, iNOS and TNF-α. In aorta segments, OLE prevented endothelial dysfunction and vascular insulin resistance and improved vasoconstriction in response to KCl and NA. Improvement in vascular function was associated with the attenuation of the alterations in the gene expression of COX-2, IL-6, GPx, NOX-1 and IL-10. In conclusion, OLE exerts anti-inflammatory and antioxidant effects in aged rats and attenuates the alterations in vascular function associated with aging.

Molecular Imaging of Infarcted Heart by Biofunctionalized Gold Nanoshells.

The unique combination of physical and optical properties of silica (core)/gold (shell) nanoparticles (gold nanoshells) makes them especially suitable for biomedicine. Gold nanoshells are used from high-resolution in vivo imaging to in vivo photothermal tumor treatment. Furthermore, their large scattering cross-section in the second biological window (1000-1700 nm) makes them also especially adequate for molecular optical coherence tomography (OCT). In this work, it is demonstrated that, after suitable functionalization, gold nanoshells in combination with clinical OCT systems are capable of imaging damage in the myocardium following an infarct. Since both inflammation and apoptosis are two of the main mechanisms underlying myocardial damage after ischemia, such damage imaging is achieved by endowing gold nanoshells with selective affinity for the inflammatory marker intercellular adhesion molecule 1 (ICAM-1), and the apoptotic marker phosphatidylserine. The results here presented constitute a first step toward a fast, safe, and accurate diagnosis of damaged tissue within infarcted hearts at the molecular level by means of the highly sensitive OCT interferometric technique.

Beneficial Effects of a Mixture of Algae and Extra Virgin Olive Oils on the Age-Induced Alterations of Rodent Skeletal Muscle: Role of HDAC-4.

Aging is associated with a progressive decline in skeletal muscle mass, strength and function (sarcopenia). We have investigated whether a mixture of algae oil (25%) and extra virgin olive oil (75%) could exert beneficial effects on sarcopenia. Young (3 months) and old (24 months) male Wistar rats were treated with vehicle or with the oil mixture (OM) (2.5 mL/kg) for 21 days. Aging decreased gastrocnemius weight, total protein, and myosin heavy chain mRNA. Treatment with the OM prevented these effects. Concomitantly, OM administration decreased the inflammatory state in muscle; it prevented the increase of pro-inflammatory interleukin-6 (IL-6) and the decrease in anti-inflammatory interleukin-10 (IL-10) in aged rats. The OM was not able to prevent aging-induced alterations in either the insulin-like growth factor I/protein kinase B (IGF-I/Akt) pathway or in the increased expression of atrogenes in the gastrocnemius. However, the OM prevented decreased autophagy activity (ratio protein 1A/1B-light chain 3 (LC3b) II/I) induced by aging and increased expression of factors related with muscle senescence such as histone deacetylase 4 (HDAC-4), myogenin, and IGF-I binding protein 5 (IGFBP-5). These data suggest that the beneficial effects of the OM on muscle can be secondary to its anti-inflammatory effect and to the normalization of HDAC-4 and myogenin levels, making this treatment an alternative therapeutic tool for sarcopenia.

A Mixture of Algae and Extra Virgin Olive Oils Attenuates the Cardiometabolic Alterations Associated with Aging in Male Wistar Rats.

Aging is one of the major risk factors for suffering cardiovascular and metabolic diseases. Due to the increase in life expectancy, there is a strong interest in the search for anti-aging strategies to treat and prevent these aging-induced disorders. Both omega 3 polyunsaturated fatty acids (ω-3 PUFA) and extra virgin olive oil (EVOO) exert numerous metabolic and cardiovascular benefits in the elderly. In addition, EVOO constitutes an interesting ingredient to stabilize ω-3 PUFA and decrease their oxidation process due to its high content in antioxidant compounds. ω-3 PUFA are commonly obtained from fish. However, more ecological and sustainable sources, such as algae oil (AO) can also be used. In this study, we aimed to study the possible beneficial effect of an oil mixture composed by EVOO (75%) and AO (25%) rich in ω-3 PUFA (35% docosahexaenoic acid (DHA) and 20% eicosapentaenoic acid (EPA)) on the cardiometabolic alterations associated with aging. For this purpose; young (three months old) and old (24 months old) male Wistar rats were treated with vehicle or with the oil mixture (2.5 mL/kg) for 21 days. Treatment with the oil mixture prevented the aging-induced increase in the serum levels of saturated fatty acids (SFA) and the aging-induced decrease in the serum concentrations of mono-unsaturated fatty acids (MUFA). Old treated rats showed increased serum concentrations of EPA and DHA and decreased HOMA-IR index and circulating levels of total cholesterol, insulin and IL-6. Treatment with the oil mixture increased the mRNA levels of antioxidant and insulin sensitivity-related enzymes, as well as reduced the gene expression of pro-inflammatory markers in the liver and in cardiac and aortic tissues. In addition, the treatment also prevented the aging-induced endothelial dysfunction and vascular insulin resistance through activation of the PI3K/Akt pathway. Moreover, aortic rings from old rats treated with the oil mixture showed a decreased response to the vasoconstrictor AngII. In conclusion, treatment with a mixture of EVOO and AO improves the lipid profile, insulin sensitivity and vascular function in aged rats and decreases aging-induced inflammation and oxidative stress in the liver, and in the cardiovascular system. Thus, it could be an interesting strategy to deal with cardiometabolic alterations associated with aging.

Instantaneous In Vivo Imaging of Acute Myocardial Infarct by NIR-II Luminescent Nanodots.

Fast and precise localization of ischemic tissues in the myocardium after an acute infarct is required by clinicians as the first step toward accurate and efficient treatment. Nowadays, diagnosis of a heart attack at early times is based on biochemical blood analysis (detection of cardiac enzymes) or by ultrasound-assisted imaging. Alternative approaches are investigated to overcome the limitations of these classical techniques (time-consuming procedures or low spatial resolution). As occurs in many other fields of biomedicine, cardiological preclinical imaging can also benefit from the fast development of nanotechnology. Indeed, bio-functionalized near-infrared-emitting nanoparticles are herein used for in vivo imaging of the heart after an acute myocardial infarct. Taking advantage of the superior acquisition speed of near-infrared fluorescence imaging, and of the efficient selective targeting of the near-infrared-emitting nanoparticles, in vivo images of the infarcted heart are obtained only a few minutes after the acute infarction event. This work opens an avenue toward cost-effective, fast, and accurate in vivo imaging of the ischemic myocardium after an acute infarct.

Supplementation with a Carob (Ceratonia siliqua L.) Fruit Extract Attenuates the Cardiometabolic Alterations Associated with Metabolic Syndrome in Mice.

The incidence of metabolic syndrome (MetS) is increasing worldwide which makes necessary the finding of new strategies to treat and/or prevent it. The aim of this study was to analyze the possible beneficial effects of a carob fruit extract (CSAT+®) on the cardiometabolic alterations associated with MetS in mice. 16-week-old C57BL/6J male mice were fed for 26 weeks either with a standard diet (chow) or with a diet rich in fats and sugars (HFHS), supplemented or not with 4.8% of CSAT+®. CSAT+® supplementation reduced blood glucose, Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and circulating levels of total cholesterol, low-density lipoprotein (LDL) cholesterol (LDL-c), insulin, and interleukin-6 (IL-6). In adipose tissue and skeletal muscle, CSAT+® prevented MetS-induced insulin resistance, reduced macrophage infiltration and the expression of pro-inflammatory markers, and up-regulated the mRNA levels of antioxidant markers. Supplementation with CSAT+® prevented MetS-induced hypertension and decreased the vascular response of aortic rings to angiotensin II (AngII). Moreover, treatment with CSAT+® attenuated endothelial dysfunction and increased vascular sensitivity to insulin. In the heart, CSAT+® supplementation reduced cardiomyocyte apoptosis and prevented ischemia-reperfusion-induced decrease in cardiac contractility. The beneficial effects at the cardiovascular level were associated with a lower expression of pro-inflammatory and pro-oxidant markers in aortic and cardiac tissues.

Overfeeding During Lactation in Rats is Associated with Cardiovascular Insulin Resistance in the Short-Term.

Childhood obesity is associated with metabolic and cardiovascular comorbidities. The development of these alterations may have its origin in early life stages such as the lactation period through metabolic programming. Insulin resistance is a common complication in obese patients and may be responsible for the cardiovascular alterations associated with this condition. This study analyzed the development of cardiovascular insulin resistance in a rat model of childhood overweight induced by overfeeding during the lactation period. On birth day, litters were divided into twelve (L12) or three pups per mother (L3). Overfed rats showed a lower increase in myocardial contractility in response to insulin perfusion and a reduced insulin-induced vasodilation, suggesting a state of cardiovascular insulin resistance. Vascular insulin resistance was due to decreased activation of phosphoinositide 3-kinase (PI3K)/Akt pathway, whereas cardiac insulin resistance was associated with mitogen-activated protein kinase (MAPK) hyperactivity. Early overfeeding was also associated with a proinflammatory and pro-oxidant state; endothelial dysfunction; decreased release of nitrites and nitrates; and decreased gene expression of insulin receptor (IR), glucose transporter-4 (GLUT-4), and endothelial nitric oxide synthase (eNOS) in response to insulin. In conclusion, overweight induced by lactational overnutrition in rat pups is associated with cardiovascular insulin resistance that could be related to the cardiovascular alterations associated with this condition.

Spontaneous Pulmonary Hypertension Associated With Systemic Sclerosis in P-Selectin Glycoprotein Ligand 1-Deficient Mice.

OBJECTIVE: Pulmonary arterial hypertension (PAH), one of the major complications of systemic sclerosis (SSc), is a rare disease with unknown etiopathogenesis and noncurative treatments. As mice deficient in P-selectin glycoprotein ligand 1 (PSGL-1) develop a spontaneous SSc-like syndrome, we undertook this study to analyze whether they develop PAH and to examine the molecular mechanisms involved. METHODS: Doppler echocardiography was used to estimate pulmonary pressure, immunohistochemistry was used to assess vascular remodeling, and myography of dissected pulmonary artery rings was used to analyze vascular reactivity. Angiotensin II (Ang II) levels were quantified by enzyme-linked immunosorbent assay, and Western blotting was used to measure Ang II type 1 receptor (AT1 R), AT2 R, endothelial cell nitric oxide synthase (eNOS), and phosphorylated eNOS expression in lung lysates. Flow cytometry allowed us to determine cytokine production by immune cells and NO production by endothelial cells. In all cases, there were 4-8 mice per experimental group. RESULTS: PSGL-1-/- mice showed lung vessel wall remodeling and a reduced mean ± SD expression of pulmonary AT2 R (expression ratio [relative to ß-actin] in female mice age >18 months: wild-type mice 0.799 ± 0.508 versus knockout mice 0.346 ± 0.229). With aging, female PSGL-1-/- mice had impaired up-regulation of estrogen receptor α (ERα) and developed lung vascular endothelial dysfunction coinciding with an increase in mean ± SEM pulmonary Ang II levels (wild-type 48.70 ± 5.13 pg/gm lung tissue versus knockout 78.02 ± 28.09 pg/gm lung tissue) and a decrease in eNOS phosphorylation, leading to reduced endothelial NO production. These events led to a reduction in the pulmonary artery acceleration time:ejection time ratio in 33% of aged female PSGL-1-/- mice, indicating pulmonary hypertension. Importantly, we found expanded populations of interferon-γ-producing PSGL-1-/- T cells and B cells and a reduced presence of regulatory T cells. CONCLUSION: The absence of PSGL-1 induces a reduction in Treg cells, NO production, and ERα expression and causes an increase in Ang II in the lungs of female mice, favoring the development of PAH.

P-Selectin preserves immune tolerance in mice and is reduced in human cutaneous lupus.

Mice deficient in P-Selectin presented altered immunity/tolerance balance. We have observed that the absence of P-Selectin promotes splenomegaly with reduced naïve T cell population, elevated activated/effector T cell subset, increased germinal center B and Tfh populations and high production of autoreactive antibodies. Moreover, 1.5-3-month-old P-selectin KO mice showed reduced IL-10-producing leukocytes in blood and a slightly reduced Treg population in the skin. With aging and, coinciding with disease severity, there is an increase in the IL17+ circulating and dermal T cell subpopulations and reduction of dermal Treg. As a consequence, P-Selectin deficient mice developed a progressive autoimmune syndrome showing skin alterations characteristic of lupus prone mice and elevated circulating autoantibodies, including anti-dsDNA. Similar to human SLE, disease pathogenesis was characterized by deposition of immune complexes in the dermoepidermal junction and renal glomeruli, and a complex pattern of autoantibodies. More important, skin biopsies of cutaneous lupus erythematosus patients did not show increased expression of P-Selectin, as described for other inflammatory diseases, and the number of vessels expressing P-Selectin was reduced.