Endothelial cell-specific mineralocorticoid receptor activation promotes diastolic dysfunction in diet-induced obese male mice.

Widespread consumption of diets high in fat and fructose (Western diet, WD) has led to increased prevalence of obesity and diastolic dysfunction (DD). DD is a prominent feature of heart failure with preserved ejection fraction (HFpEF). However, the underlying mechanisms of DD are poorly understood, and treatment options are still limited. We have previously shown that deletion of the cell-specific mineralocorticoid receptor in endothelial cells (ECMR) abrogates DD induced by WD feeding in female mice. However, the specific role of ECMR activation in the pathogenesis of DD in male mice has not been clarified. Therefore, we fed 4-wk-old ECMR knockout (ECMRKO) male mice and littermates (LM) with either a WD or chow diet (CD) for 16 wk. WD feeding resulted in DD characterized by increased left ventricle (LV) filling pressure (E/e') and diastolic stiffness [E/e'/LV inner diameter at end diastole (LVIDd)]. Compared with CD, WD in LM resulted in increased myocardial macrophage infiltration, oxidative stress, and increased myocardial phosphorylation of Akt, in concert with decreased phospholamban phosphorylation. WD also resulted in focal cardiomyocyte remodeling, characterized by areas of sarcomeric disorganization, loss of mitochondrial electron density, and mitochondrial fragmentation. Conversely, WD-induced DD and associated biochemical and structural abnormalities were prevented by ECMR deletion. In contrast with our previously reported observations in females, WD-fed male mice exhibited enhanced Akt signaling and a lower magnitude of cardiac injury. Collectively, our data support a critical role for ECMR in obesity-induced DD and suggest critical mechanistic differences in the genesis of DD between males and females.

AT2R agonist NP-6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm.

Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP-6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP-6A4-induced AT2R activation could mitigate AngII-induced abdominal aortic aneurism (AAA) using AngII-treated Apoe-/- mice. Male Apoe-/- mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre-treated subcutaneously with NP-6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP-6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP-6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII-induced increases in monocyte chemotactic protein-1, osteopontin and proteolytic activity of the aorta. However, NP-6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.

DAPT, a potent Notch inhibitor regresses actively growing abdominal aortic aneurysm via divergent pathways.

Abdominal aortic aneurysm (AAA) is a localized pathological dilation of the aorta exceeding the normal diameter (∼20 mm) by more than 50% of its original size (≥30 mm), accounting for approximately 150000-200000 deaths worldwide per year. We previously reported that Notch inhibition does not decrease the size of pre-established AAA at late stage of the disease. Here, we examined whether a potent pharmacologic inhibitor of Notch signaling (DAPT (N-[N-(3,5-Difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester)), regresses an actively growing AAA. In a mouse model of an aneurysm (Apoe-/- mice; n=44); DAPT (n=17) or vehicle (n=17) was randomly administered at day 14 of angiotensin II (AngII; 1 µg/min/kg), three times a week and mice were killed on day 42. Progressive increase in aortic stiffness and maximal intraluminal diameter (MILD) was observed in the AngII + vehicle group, which was significantly prevented by DAPT (P<0.01). The regression of aneurysm with DAPT was associated with reduced F4/80+Cd68+ (cluster of differentiation 68) inflammatory macrophages. DAPT improved structural integrity of aorta by reducing collagen fibrils abnormality and restoring their diameter. Mechanistically, C-C chemokine receptor type 7 (Ccr7)+F4/80- dendritic cells (DCs), implicated in the regression of aneurysm, were increased in the aorta of DAPT-treated mice. In the macrophages stimulated with AngII or lipopolysaccharide (LPS), DAPT reverted the expression of pro-inflammatory genes Il6 and Il12 back to baseline within 6 h compared with vehicle (P<0.05). DAPT also significantly increased the expression of anti-inflammatory genes, including c-Myc, Egr2, and Arg1 at 12-24 h in the LPS-stimulated macrophages (P<0.05). Overall, these regressive effects of Notch signaling inhibitor emphasize its therapeutic implications to prevent the progression of active AAAs.

Deficiency of IL12p40 (Interleukin 12 p40) Promotes Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysm.

Objective- Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40-/- (interleukin 12 p40) mice, we investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm. Approach and Results- Male (8-10 week-old) wild-type and IL12p40-/- mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40-/- mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40-/- mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented the Tgfß2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40-/- mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions- IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfß2.

MicroRNA miR145 regulates TGFBR2 expression and matrix synthesis in vascular smooth muscle cells.

RATIONALE: MicroRNA miR145 has been implicated in vascular smooth muscle cell differentiation, but its mechanisms of action and downstream targets have not been fully defined. OBJECTIVE: Here, we sought to explore and define the mechanisms of miR145 function in smooth muscle cells. METHODS AND RESULTS: Using a combination of cell culture assays and in vivo mouse models to modulate miR145, we characterized its downstream actions on smooth muscle phenotypes. Our results show that the miR-143/145 gene cluster is induced in smooth muscle cells by coculture with endothelial cells. Endothelial cell-induced expression of miR-143/145 is augmented by Notch signaling and accordingly expression is reduced in Notch receptor-deficient cells. Screens to identify miR145-regulated genes revealed that the transforming growth factor (TGF)-ß pathway has a significantly high number of putative target genes, and we show that TGFß receptor II is a direct target of miR145. Extracellular matrix genes that are regulated by TGFß receptor II were attenuated by miR145 overexpression, and miR145 mutant mice exhibit an increase in extracellular matrix synthesis. Furthermore, activation of TGFß signaling via angiotensin II infusion revealed a pronounced fibrotic response in the absence of miR145. CONCLUSIONS: These data demonstrate a specific role for miR145 in the regulation of matrix gene expression in smooth muscle cells and suggest that miR145 acts to suppress TGFß-dependent extracellular matrix accumulation and fibrosis, while promoting TGFß-induced smooth muscle cell differentiation. Our findings offer evidence to explain how TGFß signaling exhibits distinct downstream actions via its regulation by a specific microRNA.

Endothelial nitric oxide signaling regulates Notch1 in aortic valve disease.

The mature aortic valve is composed of a structured trilaminar extracellular matrix that is interspersed with aortic valve interstitial cells (AVICs) and covered by endothelium. Dysfunction of the valvular endothelium initiates calcification of neighboring AVICs leading to calcific aortic valve disease (CAVD). The molecular mechanism by which endothelial cells communicate with AVICs and cause disease is not well understood. Using a co-culture assay, we show that endothelial cells secrete a signal to inhibit calcification of AVICs. Gain or loss of nitric oxide (NO) prevents or accelerates calcification of AVICs, respectively, suggesting that the endothelial cell-derived signal is NO. Overexpression of Notch1, which is genetically linked to human CAVD, retards the calcification of AVICs that occurs with NO inhibition. In AVICs, NO regulates the expression of Hey1, a downstream target of Notch1, and alters nuclear localization of Notch1 intracellular domain. Finally, Notch1 and NOS3 (endothelial NO synthase) display an in vivo genetic interaction critical for proper valve morphogenesis and the development of aortic valve disease. Our data suggests that endothelial cell-derived NO is a regulator of Notch1 signaling in AVICs in the development of the aortic valve and adult aortic valve disease.

Inhibition of Notch1 signaling reduces abdominal aortic aneurysm in mice by attenuating macrophage-mediated inflammation.

OBJECTIVE: Activation of inflammatory pathways plays a critical role in the development of abdominal aortic aneurysms (AAA). Notch1 signaling is a significant regulator of the inflammatory response; however, its role in AAA is unknown. METHODS AND RESULTS: In an angiotensin II-induced mouse model of AAA, activation of Notch1 signaling was observed in the aortic aneurysmal tissue of Apoe(-/-) mice, and a similar activation of Notch1 was observed in aneurysms of humans undergoing AAA repair. Notch1 haploinsufficiency significantly reduced the incidence of AAA in Apoe(-/-) mice in response to angiotensin II. Reconstitution of bone marrow-derived cells from Notch1(+/-);Apoe(-/-) mice (donor) in lethally irradiated Apoe(-/-) mice (recipient) decreased the occurrence of aneurysm. Flow cytometry and immunohistochemistry demonstrated that Notch1 haploinsufficiency prevented the influx of inflammatory macrophages at the aneurysmal site by causing defects in macrophage migration and proliferation. In addition, there was an overall reduction in the inflammatory burden in the aorta of the Notch1(+/-);Apoe(-/-) mice compared with the Apoe(-/-) mice. Last, pharmacological inhibition of Notch1 signaling also prevented AAA formation and progression in Apoe(-/-) mice. CONCLUSIONS: Our data suggest that decreased levels of Notch1 protect against the formation of AAA by preventing macrophage recruitment and attenuating the inflammatory response in the aorta.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

Macrophages are essential for skeletal muscle homeostasis, but how their dysregulation contributes to the development of fibrosis in muscle disease remains unclear. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six clusters and unexpectedly found that none corresponded to traditional definitions of M1 or M2 macrophages. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 (gal-3) and osteopontin (Spp1). Spatial transcriptomics, computational inferences of intercellular communication, and in vitro assays indicated that macrophage-derived Spp1 regulates stromal progenitor differentiation. Gal-3+ macrophages were chronically activated in dystrophic muscle, and adoptive transfer assays showed that the gal-3+ phenotype was the dominant molecular program induced within the dystrophic milieu. Gal-3+ macrophages were also elevated in multiple human myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining their transcriptional programs and reveal Spp1 as a major regulator of macrophage and stromal progenitor interactions.

Single-cell and spatial transcriptomics identify a macrophage population associated with skeletal muscle fibrosis.

The monocytic/macrophage system is essential for skeletal muscle homeostasis, but its dysregulation contributes to the pathogenesis of muscle degenerative disorders. Despite our increasing knowledge of the role of macrophages in degenerative disease, it still remains unclear how macrophages contribute to muscle fibrosis. Here, we used single-cell transcriptomics to determine the molecular attributes of dystrophic and healthy muscle macrophages. We identified six novel clusters. Unexpectedly, none corresponded to traditional definitions of M1 or M2 macrophage activation. Rather, the predominant macrophage signature in dystrophic muscle was characterized by high expression of fibrotic factors, galectin-3 and spp1. Spatial transcriptomics and computational inferences of intercellular communication indicated that spp1 regulates stromal progenitor and macrophage interactions during muscular dystrophy. Galectin-3 + macrophages were chronically activated in dystrophic muscle and adoptive transfer assays showed that the galectin-3 + phenotype was the dominant molecular program induced within the dystrophic milieu. Histological examination of human muscle biopsies revealed that galectin-3 + macrophages were also elevated in multiple myopathies. These studies advance our understanding of macrophages in muscular dystrophy by defining the transcriptional programs induced in muscle macrophages, and reveal spp1 as a major regulator of macrophage and stromal progenitor interactions.

Requirement for inducible nitric oxide synthase in chronic allergen exposure-induced pulmonary fibrosis but not inflammation.

The role of inducible NO synthase (iNOS) in allergic airway inflammation remains elusive. We tested the hypothesis that iNOS plays different roles during acute versus chronic airway inflammation. Acute and chronic mouse models of OVA-induced airway inflammation were used to conduct the study. We showed that iNOS deletion was associated with a reduction in eosinophilia, mucus hypersecretion, and IL-5 and IL-13 production upon the acute protocol. Such protection was completely abolished upon the chronic protocol. Interestingly, pulmonary fibrosis observed in wild-type mice under the chronic protocol was completely absent in iNOS(-/-) mice despite persistent IL-5 and IL-13 production, suggesting that these cytokines were insufficient for pulmonary fibrosis. Such protection was associated with reduced collagen synthesis and indirect but severe TGF-beta modulation as confirmed using primary lung smooth muscle cells. Although activation of matrix metalloproteinase-2/-9 exhibited little change, the large tissue inhibitor of metalloproteinase-2 (TIMP-2) increase detected in wild-type mice was absent in the iNOS(-/-) counterparts. The regulatory effect of iNOS on TIMP-2 may be mediated by peroxynitrite, as the latter reversed TIMP-2 expression in iNOS(-/-) lung smooth muscle cells and fibroblasts, suggesting that the iNOS-TIMP-2 link may explain the protective effect of iNOS-knockout against pulmonary fibrosis. Analysis of lung sections from chronically OVA-exposed iNOS(-/-) mice revealed evidence of residual but significant protein nitration, prevalent oxidative DNA damage, and poly(ADP-ribose) polymerase-1 activation. Such tissue damage, inflammatory cell recruitment, and mucus hypersecretion may be associated with substantial arginase expression and activity. The results in this study exemplify the complexity of the role of iNOS in asthma and the preservation of its potential as a therapeutic a target.

Poly(ADP-ribose) polymerase-1 is a determining factor in Crm1-mediated nuclear export and retention of p65 NF-kappa B upon TLR4 stimulation.

The role of NF-kappaB in the expression of inflammatory genes and its participation in the overall inflammatory process of chronic diseases and acute tissue injury are well established. We and others have demonstrated a critical involvement of poly(ADP-ribose) polymerase (PARP)-1 during inflammation, in part, through its relationship with NF-kappaB. However, the mechanism by which PARP-1 affects NF-kappaB activation has been elusive. In this study, we show that PARP-1 inhibition by gene knockout, knockdown, or pharmacologic blockade prevented p65 NF-kappaB nuclear translocation in smooth muscle cells upon TLR4 stimulation, NF-kappaB DNA-binding activity, and subsequent inducible NO synthase and ICAM-1 expression. Such defects were reversed by reconstitution of PARP-1 expression. PARP-1 was dispensable for LPS-induced IkappaBalpha phosphorylation and subsequent degradation but was required for p65 NF-kappaB phosphorylation. A perinuclear p65 NF-kappaB localization in LPS-treated PARP-1(-/-) cells was associated with an export rather an import defect. Indeed, whereas PARP-1 deficiency did not alter expression of importin alpha3 and importin alpha4 and their cytosolic localization, the cytosolic levels of exportin (Crm)-1 were increased. Crm1 inhibition promoted p65 NF-kappaB nuclear accumulation as well as reversed LPS-induced p65 NF-kappaB phosphorylation and inducible NO synthase and ICAM-1 expression. Interestingly, p65 NF-kappaB poly(ADP-ribosyl)ation decreased its interaction with Crm1 in vitro. Pharmacologic inhibition of PARP-1 increased p65 NF-kappaB-Crm1 interaction in LPS-treated smooth muscle cells. These results suggest that p65 NF-kappaB poly(ADP-ribosyl)ation may be a critical determinant for the interaction with Crm1 and its nuclear retention upon TLR4 stimulation. These results provide novel insights into the mechanism by which PARP-1 promotes NF-kappaB nuclear retention, which ultimately can influence NF-kappaB-dependent gene regulation.

Structurally abnormal collagen fibrils in abdominal aortic aneurysm resist platelet adhesion.

BACKGROUND: Platelet adhesion to the subendothelial collagen fibrils is one of the first steps in hemostasis. Understanding how structural perturbations in the collagen fibril affect platelet adhesion can provide novel insights into disruption of hemostasis in various diseases. We have recently identified the presence of abnormal collagen fibrils with compromised D-periodic banding in the extracellular matrix remodeling present in abdominal aortic aneurysms (AAA). OBJECTIVE: In this study, we employed multimodal microscopy approaches to characterize how collagen fibril structure impacts platelet adhesion in clinical AAA tissues. METHODS: Ultrastructural atomic force microscopy (AFM) analysis was performed on tissue sections after staining with fluorescently labeled collagen hybridizing peptide (CHP) to recognize degraded collagen. Second harmonic generation (SHG) microscopy was used on CHP-stained sections to identify regions of intact versus degraded collagen. Finally, platelet adhesion was identified via SHG and indirect immunofluorescence on the same tissue sections. RESULTS: Our results indicate that ultrastructural features characterizing collagen fibril abnormalities coincide with CHP staining. SHG signal was absent from CHP-positive regions. Additionally, platelet binding was primarily localized to regions with SHG signal. Abnormal collagen fibrils present in AAA (in SHG negative regions) were thus found to inhibit platelet adhesion compared to normal fibrils. CONCLUSIONS: Our investigations reveal how the collagen fibril structure in the vessel wall can serve as another regulator of platelet-collagen adhesion. These results can be broadly applied to understand the role of collagen fibril structure in regulating thrombosis or bleeding disorders.

Siddha fasting in obese acute decompensated heart failure may improve hospital outcomes through empowerment and natural ketosis.

BACKGROUND: Morbid obesity (BMI > 35 kg/m2 with comorbid conditions) is present in 25 - 35% of acute decompensated heart failure (AHF) patients. Prevalence of HF increases with duration of morbid obesity from 30% at 15 years to over 90% at 30 years. There is a need to develop pragmatic therapies that address the unique physical and mental challenges faced by obese AHF patients. Siddha is 5,000 year old Tamil Medicine using yoga and mind-body methods towards higher consciousness. Hunger gratitude Experience (HUGE) is intuitive Siddha fasting method which may improve in-hospital AHF outcomes independent of weight reduction. CASE SUMMARY: We present 5 cases of morbidly obese patients with cardiorenal syndrome (CRS) that began intermittent fasting either during their AHF hospitalization or in the outpatient setting for refractory symptoms despite hospitalization. Initiation of fasting correlated with reduction of respiratory distress and edema as well as improvements in psychological wellbeing and functional capacity. DISCUSSION: Siddha fasting mediates hemodynamic and anti-inflammatory effects through natural ketosis and psychological benefits through empowerment in AHF. Potential role of fasting in reducing myocardial workload, coronary steal, angina, volume overload, and CRS needs further study in cardiac patients.

Interleukin 12p40 Deficiency Promotes Abdominal Aortic Aneurysm by Activating CCN2/MMP2 Pathways.

Background Development of abdominal aortic aneurysm (AAA) is associated with proinflammatory cytokines including interleukin-12 (IL12). Deficiency of interleukin 12p40 (IL12p40) increases localized fibrotic events by promoting TGFß2 (transforming growth factor ß)-dependent anti-inflammatory response. Here, we determined whether IL12p40 deficiency in apolipoprotein E-/- mice attenuates the development of AAA by antagonizing proinflammatory response. Methods and Results Double knockout (DKO) mice were generated by crossbreeding IL12p40-/- mice with apolipoprotein E-/- mice (n=12). Aneurysmal studies were performed using angiotensin II (1 µg/kg/min; subcutaneous). Surprisingly, DKO mice did not prevent the development of AAA with angiotensin II infusion. Immunohistological analysis, however, showed distinct pathological features between apolipoprotein E-/- and DKO mice. Polymerase chain reaction (7 day) and cytokine arrays (28 day) of the aortic tissues from DKO mice showed significantly increased expression of cytokines related to anti-inflammatory response (interleukin 5 and interleukin 13), synthetic vascular smooth muscle cell phenotype (Activin receptor-like kinase-1 (ALK-1), artemin, and betacellulin) and T helper 17-associated response (4-1BB, interleukin-17e (Il17e) and Cd40 ligand (Cd-40L)). Indeed, DKO mice exhibited increased expression of the fibro-proteolytic pathway in the medial layer of aortae induced by cellular communication network factor 2 (CCN2) and Cd3+IL17+ cells compared with apolipoprotein E-/- mice. Laser capture microdissection showed predominant expression of CCN2/TGFß2 in the medial layer of human AAA. Finally, Ccn2 haploinsufficiency in the mice showed decreased AAA incidence in response to elastase infusion, associated with decreased matrix metalloproteinase-2 expression. Conclusions Our study reveals novel roles for IL12p40 deficiency in inducing fibro-proteolytic activities in the aneurysmal mouse model. Mechanistically, these effects of IL12p40 deficiency are mediated by CCN2/matrix metalloproteinase-2 crosstalk in the medial layer of aneurysmal aortae.

Mathematical model of atherosclerotic aneurysm.

Atherosclerosis is a major cause of abdominal aortic aneurysm (AAA) and up to 80% of AAA patients have atherosclerosis. Therefore it is critical to understand the relationship and interactions between atherosclerosis and AAA to treat atherosclerotic aneurysm patients more effectively. In this paper, we develop a mathematical model to mimic the progression of atherosclerotic aneurysms by including both the multi-layer structured arterial wall and the pathophysiology of atherosclerotic aneurysms. The model is given by a system of partial differential equations with free boundaries. Our results reveal a 2D biomarker, the cholesterol ratio and DDR1 level, assessing the risk of atherosclerotic aneurysms. The efficacy of different treatment plans is also explored via our model and suggests that the dosage of anti-cholesterol drugs is significant to slow down the progression of atherosclerotic aneurysms while the additional anti-DDR1 injection can further reduce the risk.

Green nanotechnology of MGF-AuNPs for immunomodulatory intervention in prostate cancer therapy.

Men with castration-resistant prostate cancer (CRPC) face poor prognosis and increased risk of treatment-incurred adverse effects resulting in one of the highest mortalities among patient population globally. Immune cells act as double-edged sword depending on the tumor microenvironment, which leads to increased infiltration of pro-tumor (M2) macrophages. Development of new immunomodulatory therapeutic agents capable of targeting the tumor microenvironment, and hence orchestrating the transformation of pro-tumor M2 macrophages to anti-tumor M1, would substantially improve treatment outcomes of CRPC patients. We report, herein, Mangiferin functionalized gold nanoparticulate agent (MGF-AuNPs) and its immunomodulatory characteristics in treating prostate cancer. We provide evidence of immunomodulatory intervention of MGF-AuNPs in prostate cancers through observations of enhanced levels of anti-tumor cytokines (IL-12 and TNF-α) with concomitant reductions in the levels of pro-tumor cytokines (IL-10 and IL-6). In the MGF-AuNPs treated groups, IL-12 was elevated to ten-fold while TNF-α was elevated to about 50-fold, while IL-10 and IL-6 were reduced by two-fold. Ability of MGF-AuNPs to target splenic macrophages is invoked via targeting of NF-kB signaling pathway. Finally, therapeutic efficacy of MGF-AuNPs, in treating prostate cancer in vivo in tumor bearing mice, is described taking into consideration various immunomodulatory interventions triggered by this green nanotechnology-based nanomedicine agent.

Mineralocorticoid Receptor in Myeloid Cells Mediates Angiotensin II-Induced Vascular Dysfunction in Female Mice.

Enhanced mineralocorticoid receptor (MR) signaling is critical to the development of endothelial dysfunction and arterial stiffening. However, there is a lack of knowledge about the role of MR-induced adipose tissue inflammation in the genesis of vascular dysfunction in women. In this study, we hypothesize that MR activation in myeloid cells contributes to angiotensin II (Ang II)-induced aortic stiffening and endothelial dysfunction in females via increased pro-inflammatory (M1) macrophage polarization. Female mice lacking MR in myeloid cells (MyMRKO) were infused with Ang II (500 ng/kg/min) for 4 weeks. This was followed by determinations of aortic stiffness and vasomotor responses, as well as measurements of markers of inflammation and macrophage infiltration/polarization in different adipose tissue compartments. MyMRKO mice were protected against Ang II-induced aortic endothelial stiffening, as assessed via atomic force microscopy in aortic explants, and vasorelaxation dysfunction, as measured by aortic wire myography. In alignment, MyMRKO mice were protected against Ang II-induced macrophage infiltration and M1 polarization in visceral adipose tissue (VAT) and thoracic perivascular adipose tissue (tPVAT). Collectively, this study demonstrates a critical role of MR activation in myeloid cells in the pathogenesis of vascular dysfunction in females associated with pro-inflammatory macrophage polarization in VAT and tPVAT. Our data have potential clinical implications for the prevention and management of cardiovascular disease in women, who are disproportionally at higher risk for poor outcomes.

Measurement of Pulse Propagation Velocity, Distensibility and Strain in an Abdominal Aortic Aneurysm Mouse Model.

An abdominal aortic aneurysm (AAA) is defined as a localized dilation of the abdominal aorta that exceeds the maximal intraluminal diameter (MILD) by 1.5 times of its original size. Clinical and experimental studies have shown that small aneurysms may rupture, while a subpopulation of large aneurysms may remain stable. Thus, in addition to the measurement of intraluminal diameter of the aorta, knowledge of structural traits of the vessel wall may provide important information to assess the stability of the AAA. Aortic stiffening has recently emerged as a reliable tool to determine early changes in the vascular wall. Pulse propagation velocity (PPV) along with the distensibility and radial strain are highly useful ultrasound-based methods relevant for assessing aortic stiffness. The primary purpose of this protocol is to provide a comprehensive technique for the use of ultrasound imaging system to acquire images and analyze the structural and functional properties of the aorta as determined by MILD, PPV, distensibility and radial strain.

Collagen fibril abnormalities in human and mice abdominal aortic aneurysm.

Vascular diseases like abdominal aortic aneurysms (AAA) are characterized by a drastic remodeling of the vessel wall, accompanied with changes in the elastin and collagen content. At the macromolecular level, the elastin fibers in AAA have been reported to undergo significant structural alterations. While the undulations (waviness) of the collagen fibers is also reduced in AAA, very little is understood about changes in the collagen fibril at the sub-fiber level in AAA as well as in other vascular pathologies. In this study we investigated structural changes in collagen fibrils in human AAA tissue extracted at the time of vascular surgery and in aorta extracted from angiotensin II (AngII) infused ApoE-/- mouse model of AAA. Collagen fibril structure was examined using transmission electron microscopy and atomic force microscopy. Images were analyzed to ascertain length and depth of D-periodicity, fibril diameter and fibril curvature. Abnormal collagen fibrils with compromised D-periodic banding were observed in the excised human tissue and in remodeled regions of AAA in AngII infused mice. These abnormal fibrils were characterized by statistically significant reduction in depths of D-periods and an increased curvature of collagen fibrils. These features were more pronounced in human AAA as compared to murine samples. Thoracic aorta from Ang II-infused mice, abdominal aorta from saline-infused mice, and abdominal aorta from non-AAA human controls did not contain abnormal collagen fibrils. The structural alterations in abnormal collagen fibrils appear similar to those reported for collagen fibrils subjected to mechanical overload or chronic inflammation in other tissues. Detection of abnormal collagen could be utilized to better understand the functional properties of the underlying extracellular matrix in vascular as well as other pathologies. STATEMENT OF SIGNIFICANCE: Several vascular diseases including abdominal aortic aneurysm (AAA) are characterized by extensive remodeling in the vessel wall. Although structural alterations in elastin fibers are well characterized in vascular diseases, very little is known about the collagen fibril structure in these diseases. We report here a comprehensive ultrastructural evaluation of the collagen fibrils in AAA, using high-resolution microscopy techniques like transmission electron microscopy (TEM) and atomic force microscopy (AFM). We elucidate how abnormal collagen fibrils with compromised D-periodicity and increased fibril curvature are present in the vascular tissue in both clinical AAA as well as in murine models. We discuss how these abnormal collagen fibrils are likely a consequence of mechanical overload accompanying AAA and could impact the functional properties of the underlying tissue.

High-fat diet induces lung remodeling in ApoE-deficient mice: an association with an increase in circulatory and lung inflammatory factors.

Hypercholesterolemia is increasingly considered the basis for not only cardiovascular pathologies but also several complications affecting other organs such as lungs. In this study, we examined the effect of hypercholesterolemia on lung integrity using a mouse model (ApoE(-/-)) of high-fat (HF) diet-induced atherosclerosis. A 12-week HF diet regimen induced systemic production of TNF-alpha, IFN-gamma, GMC-SF, RANTES, IL-1alpha, IL-2 and IL-12 with TNF-alpha as the predominant cytokine in ApoE(-/-) mice. Concomitantly, TNF-alpha, IFN-gamma and MIP-1alpha were detected in brochoalveolar lavage (BAL) fluids of these mice, coinciding with lung inflammation consisting primarily of monocytes/macrophages. Such lung inflammation correlated with marked collagen deposition and an increase in matrix metalloproteinase-9 activity in ApoE(-/-)mice without mucus production. Although TGF-beta1 was undetectable in the BAL fluid of ApoE(-/-) mice on HF diet, it showed a much wider tissue distribution compared with that of control animals. Direct exposure of smooth muscle cells to oxidized-LDL, in vitro, induced a time-dependent expression of TNF-alpha. Direct intratracheal TNF-alpha-administration induced a lung inflammation pattern in wild-type mice that was strikingly similar to that induced by HF diet in ApoE(-/-) mice. TNF-alpha administration induced expression of several factors known to be critically involved in lung remodeling, such as MCP-1, IL-1beta, TGF-beta1, adhesion molecules, collagen type-I and TNF-alpha itself in the lungs of treated mice. These results suggest that hypercholesterolemia may promote chronic inflammatory conditions in lungs that are conducive to lung remodeling potentially through TNF-alpha-mediated processes.