ASSOCIATIONS BETWEEN ACCELERATED ATHEROSCLEROSIS, OXIDIZED LDL IMMUNE COMPLEXES, AND IN VITRO ENDOTHELIAL DYSFUNCTION IN SYSTEMIC LUPUS ERYTHEMATOSUS.

Systemic lupus erythematosus (SLE) is an independent risk factor for atherosclerosis. This study was designed to determine the association between atherosclerosis, oxidized LDL immune complexes (oxLDL-IC), and endothelial dysfunction in SLE. SLE patients were recruited, and carotid atherosclerotic total plaque area (TPA) was determined by ultrasound. Levels of oxLDL-IC were measured. In vitro endothelial function was measured by aortic endothelial nitric oxide (NO) production after culture of human aortic endothelial cells (HAEC) with SLE serum. Levels of oxLDL-IC are associated significantly with TPA. In vitro HAEC NO production after culture with SLE serum was positively correlated with serum complement. HAEC NO production was increased with sepiapterin to couple eNOS. To our knowledge, this is the first study to demonstrate an association between subclinical accelerated atherosclerosis and oxLDL-IC in SLE. This is also the first study to demonstrate the effect of sepiapterin on improving in vitro aortic endothelial cell function in SLE.

Role of Klotho in migration and proliferation of human dermal microvascular endothelial cells.

PURPOSE: To examine the possible role of Klotho (Kl) in human microvasculature. METHODS: The expression level of Kl in primary human dermal microvascular endothelial cells (HDMECs) and primary human dermal fibroblasts (HFb) was detected by real-time polymerase chain reaction amplification (qRT-PCR), Western blot analyses and immunohistochemistry. Migration of HDMECs and HFb was examined in monolayer wound healing "scratch assay" and Transwell assay. Proliferation of these cells was examined using Cell Proliferation BrdU incorporation assay. RESULTS: Our results have shown that downregulation of Kl abrogated HDMECs migration after 48h. On the other hand, migration of HFb significantly increased after blocking Kl. Lack of Kl decreased expression of genes involved in the activation of endothelial cells and enhanced expression of genes involved in extracellular matrix remodeling and organization of connective tissue. CONCLUSIONS: This study for the first time provides the evidence that Kl is expressed in HDMECs and HFb. Additionally, we have demonstrated that Kl is implicated in the process of angiogenesis of human dermal microvasculature.

Endothelial GATA-6 deficiency promotes pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a chronic and progressive disease characterized by pulmonary vasculopathy with elevation of pulmonary artery pressure, often culminating in right ventricular failure. GATA-6, a member of the GATA family of zinc-finger transcription factors, is highly expressed in quiescent vasculature and is frequently lost during vascular injury. We hypothesized that endothelial GATA-6 may play a critical role in the molecular mechanisms underlying endothelial cell (EC) dysfunction in PAH. Here we report that GATA-6 is markedly reduced in pulmonary ECs lining both occluded and nonoccluded vessels in patients with idiopathic and systemic sclerosis-associated PAH. GATA-6 transcripts are also rapidly decreased in rodent PAH models. Endothelial GATA-6 is a direct transcriptional regulator of genes controlling vascular tone [endothelin-1, endothelin-1 receptor type A, and endothelial nitric oxide synthase (eNOS)], pro-inflammatory genes, CX3CL1 (fractalkine), 5-lipoxygenease-activating protein, and markers of vascular remodeling, including PAI-1 and RhoB. Mice with the genetic deletion of GATA-6 in ECs (Gata6-KO) spontaneously develop elevated pulmonary artery pressure and increased vessel muscularization, and these features are further exacerbated in response to hypoxia. Furthermore, innate immune cells including macrophages (CD11b(+)/F4/80(+)), granulocytes (Ly6G(+)/CD45(+)), and dendritic cells (CD11b(+)/CD11c(+)) are significantly increased in normoxic Gata6-KO mice. Together, our findings suggest a critical role of endothelial GATA-6 deficiency in development and disease progression in PAH.

Are microparticles the missing link between thrombosis and autoimmune diseases? Involvement in selected rheumatologic diseases.

Microparticles (MPs) are membrane-bound vesicles with important physiologic effects. MPs exchange information intercellularly, with each kind of MP carrying antigens and receptors of the cells from which they originated. They are biologic effectors in inflammation, angiogenesis, vascular injury, and thrombosis. Thrombosis is generally caused by abnormalities in blood flow, blood composition, and/or properties of the vessel wall. Thrombosis is a well-described feature of cardiovascular disease and cerebrovascular disease. Accumulating evidence suggests that increased risk of thrombosis is also characteristic of autoimmune disorders and immune-mediated diseases affecting all age groups, although the older adults are most vulnerable. Current research has also implicated MPs as a source of autoantigenic nuclear material that can form immune complexes, activate the innate immune system, and may lead to autoimmunity. This review focuses on the contribution of MPs to both the pathogenesis of autoimmune diseases and, as the immune and coagulation systems are tightly linked, their role in hypercoagulability in the setting of autoimmunity in an aging population.

Connective tissue growth factor (CTGF/CCN2) mediates angiogenic effect of S1P in human dermal microvascular endothelial cells.

OBJECTIVE: The primary objective of this study was to examine the potential interaction between S1P, a pleiotropic lipid mediator, and CTGF/CCN2, a secreted multimodular protein, in the process of endothelial cell migration. The secondary objective was to determine whether C- and N-terminal domains of CTGF/CCN2 have a specific function in cell migration. MATERIALS AND METHODS: Migration of HDMECs was examined in monolayer wound healing "scratch" assay, whereas capillary-like tube formation was examined in three-dimensional collagen co-culture assays. RESULTS: We observed that S1P stimulates migration of HDMECs concomitant with upregulation of CTGF/CCN2 expression. Furthermore, the blockade of endogenous CTGF/CCN2 via siRNA abrogated S1P-induced HDMEC migration and capillary-like tube formation. Full-length CTGF induced cell migration and capillary-like tube formation with a potency similar to that of S1P, while C-terminal domain of CTGF was slightly less effective. However, N-terminal domain had only a residual activity in inducing capillary-like tube formation. CONCLUSIONS: This study revealed that CTGF/CCN2 is required for the S1P-induced endothelial cell migration, which suggests that CTGF/CCN2 may be an important mediator of S1P-induced physiological and pathological angiogenesis. Moreover, this study shows that the pro-migratory activity of CTGF/CCN2 is located in the C-terminal domain.

Association Between the Anti-Aging Gene Klotho and Selected Rheumatologic Autoimmune Diseases.

Klotho long recognized for its role in anti-aging, is potentially implicated in the pathogenesis of rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis. Aging of the immune system coincides with the inability of the body to recognize self-antigens, which often leads to autoimmune responses. The role of Klotho in these autoimmune diseases should be of high interest; however, few articles have been published exploring the role of Klotho in the pathogenesis, organ involvement, or clinical manifestation of rheumatoid arthritis, systemic lupus erythematosus, and systemic sclerosis. Herein, we discuss information gathered from peer-reviewed publications to describe the emerging role of Kl in these select rheumatologic autoimmune diseases.

Lupus serum induces inflammatory interaction with neutrophils in human glomerular endothelial cells.

OBJECTIVES: SLE is associated with endothelial cell dysfunction (ECD). Understanding how ECD leads to neutrophil infiltration into glomeruli is essential to finding therapeutic targets for SLE. The aim of this study is to determine the effect of SLE serum from patients with active disease to induce neutrophil adhesion to and chemotaxis towards glomerular endothelial cells and factors induced by serum that associate with neutrophil chemotaxis. METHODS: Patients with SLE had serum collected during paired longitudinal visits with lower and higher activity. 13 patients with SLE (5 SLE, 5 SLE with hypertension (HTN) and 3 SLE lupus nephritis (LN) and HTN), and 10 healthy controls (5 with and 5 without HTN) were examined. The adhesion of neutrophils to serum-treated human renal glomerular endothelial cells (HRGECs) or chemotaxis of neutrophils towards conditioned media from serum-treated HRGECs was determined, and levels of cytokines in this conditioned medium were quantified. Pathway analysis of cytokines induced by SLE and LN serum that associated with neutrophil migration was performed. RESULTS: HRGECs treated with SLE serum induced significantly greater neutrophil chemotaxis and adhesion compared with control serum. When examining specific cohorts, SLE HTN and LN HTN promoted greater neutrophil chemotaxis than control serum, while SLE HTN and LN HTN promoted greater chemotaxis than SLE serum. Serum from active disease visits promoted neutrophil chemotaxis and adhesion over paired inactive visits. Levels of platelet-derived growth factor-BB, interleukin (IL)-15 and IL-8 secreted by SLE serum-treated HRGECs positively correlated with neutrophil chemotaxis. Pathway analysis suggested that LN serum induced pathways important in endoplasmic reticulum and oxidative stress. CONCLUSIONS: SLE serum induces expression of mediators by HRGECs that promote neutrophil chemotaxis and adhesion, which increases during disease activity, and associates with factors common to pathways of endoplasmic reticulum and oxidative stress. These findings highlight the potential importance of serum factor-induced ECD in SLE and LN.

Cyr61/CCN1 and CTGF/CCN2 mediate the proangiogenic activity of VHL-mutant renal carcinoma cells.

The von Hippel-Lindau (VHL) protein serves as a negative regulator of hypoxia-inducible factor (HIF)-alpha subunits. Since HIF regulates critical angiogenic factors such as vascular endothelial growth factor (VEGF) and lesions in VHL gene are present in a majority of the highly vascularized renal cell carcinoma (RCC), it is believed that deregulation of the VHL-HIF pathway is crucial for the proangiogenic activity of RCC. Although VEGF has been confirmed as a critical angiogenic factor upregulated in VHL-mutant cells, the efficacy of antiangiogenic therapy specifically targeting VEGF signaling remains modest. In this study, we developed a three-dimensional in vitro assay to evaluate the ability of RCC cells to promote cord formation by the primary human dermal microvascular endothelial cells (HDMECs). Compared with VHL wild-type cells, VHL-mutant RCC cells demonstrated a significantly increased proangiogenic activity, which correlated with increased secretion of cysteine-rich 61 (Cyr61)/cysteine-rich 61-connective tissue growth factor-nephroblastoma overexpressed (CCN) 1, connective tissue growth factor (CTGF)/CCN2 and VEGF in conditioned culture medium. Both CCN proteins are required for HDMEC cord formation as shown by RNA interference knockdown experiments. Importantly, the proangiogenic activities conferred by the CCN proteins and VEGF are additive, suggesting non-overlapping functions. Expression of the CCN proteins is at least partly dependent on the HIF-2alpha function, the dominant HIF-alpha isoform expressed in RCC. Finally, immunohistochemical staining of Cyr61/CCN1 and CTGF/CCN2 in RCC tissue samples showed that increased expression of these proteins correlates with the loss of VHL protein expression. These findings strengthened the notion that the hypervascularized phenotype of RCC is afforded by multiple proangiogenic factors that function in parallel pathways.

Distinct effects of gonadectomy in male and female mice on collagen fibrillogenesis in the skin.

BACKGROUND: Collagen biosynthesis and deposition is a complex, multistep process, which is tightly regulated to maintain proper tissue homeostasis. Sex steroid hormones have been implicated in regulating collagen synthesis; however the specific mechanisms regulating the process remain largely unknown. OBJECTIVE: To investigate the role of estrogens and androgens in the regulation of genes involved in collagen synthesis and fibrillogenesis using gonadectomized C57/B6 mice. METHODS: Collagen content was assessed by hydroxyproline measurement and acetic acid extraction of collagen with or without the addition of pepsin. The mRNA levels of fibrillar collagens and enzymes involved in fibrillogenesis were determined by QPCR analysis. The protein expression of decorin, lumican and fibromodulin was confirmed by immunostaining. RESULTS: We have shown that castration resulted in a markedly decreased skin thickness and collagen content without affecting collagen solubility. Furthermore, the mRNA levels of fibrillar collagen genes including types I, III, and V were decreased, suggesting that androgens positively regulate the rate of collagen gene transcription. Conversely, ovariectomy mainly affected collagen solubility. The absence of estrogens resulted in decreased expression levels of several of the small leucine-rich repeat proteins and proteoglycans (SLRPs) including decorin, fibromodulin and lumican. CONCLUSIONS: Estrogens may not be directly involved in the regulation of collagen synthesis; however, they may play a critical role in regulating organization and stability of collagen fibrils. Androgens play a positive role in the regulation of collagen biosynthesis. In summary, our data demonstrate that androgens and estrogens regulate distinct aspects of collagen fibrillogenesis in mouse skin.

Impact of endothelial microparticles on coagulation, inflammation, and angiogenesis in age-related vascular diseases.

Endothelial microparticles (EMPs) are complex vesicular structures that originate from plasma membranes of activated or apoptotic endothelial cells. EMPs play a significant role in vascular function by altering the processes of inflammation, coagulation, and angiogenesis, and they are key players in the pathogenesis of several vascular diseases. Circulating EMPs are increased in many age-related vascular diseases such as coronary artery disease, peripheral vascular disease, cerebral ischemia, and congestive heart failure. Their elevation in plasma has been considered as both a biomarker and bioactive effector of vascular damage and a target for vascular diseases. This review focuses on the pleiotropic roles of EMPs and the mechanisms that trigger their formation, particularly the involvement of decreased estrogen levels, thrombin, and PAI-1 as major factors that induce EMPs in age-related vascular diseases.

A role for estrogen receptor-α and estrogen receptor-ß in collagen biosynthesis in mouse skin.

Hormonal regulation of the dermal collagenous extracellular matrix has a key role in maintaining proper tissue homeostasis. However, the factors and pathways involved in this process are not fully defined. This study investigated the role of estrogen receptors (ERs) in the regulation of collagen biosynthesis in mice lacking either ERα or ERß. Collagen content was significantly increased in the skin of ERα(-/-) mice, as measured by acetic acid extraction and the hydroxyproline assay, and correlated with the decreased levels of matrix metalloproteinase (MMP)-15 and elevated collagen production by ERα(-/-) fibroblasts. In contrast, collagen content was decreased in the skin of ERß(-/-) mice, despite markedly increased collagen production by ERß(-/-) fibroblasts. However, expression of several MMPs, including MMP-8 and -15, was significantly elevated, suggesting increased degradation of dermal collagen. Furthermore, ERß(-/-) mice were characterized by significantly reduced levels of small leucine proteoglycans, lumican (Lum), and decorin (Dcn), leading to defects in collagen fibrillogenesis and possibly less stable collagen fibrils. ERα(-/-) mice also exhibited fibrils with irregular structure and size, which correlated with increased levels of Lum and Dcn. Together, these results demonstrate distinct functions of ERs in the regulation of collagen biosynthesis in mouse skin in vivo.

Transcription factor Fli1 regulates collagen fibrillogenesis in mouse skin.

Biosynthesis of fibrillar collagen in the skin is precisely regulated to maintain proper tissue homeostasis; however, the molecular mechanisms involved in this process remain largely unknown. Transcription factor Fli1 has been shown to repress collagen synthesis in cultured dermal fibroblasts. This study investigated the role of Fli1 in regulation of collagen biosynthesis in mice skin in vivo using mice with the homozygous deletion of the C-terminal transcriptional activation (CTA) domain of the Fli1 gene (Fli1(DeltaCTA/DeltaCTA)). Skin analyses of the Fli1 mutant mice revealed a significant upregulation of fibrillar collagen genes at mRNA level, as well as increased collagen content as measured by acetic acid extraction and hydroxyproline assays. In addition, collagen fibrils contained ultrastructural abnormalities including immature thin fibrils and very thick irregularly shaped fibrils, which correlated with the reduced levels of decorin, fibromodulin, and lumican. Fibroblasts cultured from the skin of Fli1(DeltaCTA/DeltaCTA) mice maintained elevated synthesis of collagen mRNA and protein. Additional experiments in cultured fibroblasts have revealed that although Fli1 DeltaCTA retains the ability to bind to the collagen promoter in vitro and in vivo, it no longer functions as transcriptional repressor. Together, these results establish Fli1 as a key regulator of the collagen homeostasis in the skin in vivo.

Fli1 and Ets1 have distinct roles in connective tissue growth factor/CCN2 gene regulation and induction of the profibrotic gene program.

CCN2 (connective tissue growth factor), an important regulator of angiogenesis, chondrogenesis, and wound healing, is overexpressed in a majority of fibrotic diseases and in various tumors. This study investigated regulation of CCN2 gene expression by Ets family of transcription factors, focusing on two members, Fli1 and Ets1, with deregulated expression during fibrosis and tumorigenesis. We show that Ets1 and Fli1 have opposite effects on CCN2 gene expression. Ets1 functions as an activator of CCN2 transcription, whereas Fli1 acts as a repressor. A functional Ets binding site was mapped at -114 within the CCN2 promoter. This site not only mediates stimulation by Ets factors, including Ets1, Ets2, and GABPalpha/beta, but is also required for the transforming growth factor (TGF)-beta response. The contrasting functions of Ets1 and Fli1 in regulation of the CCN2 gene were confirmed by suppressing their endogenous levels using adenoviral vectors expressing specific small interfering RNAs. Additional experiments using chromatin immunoprecipitation assays have revealed that in fibroblasts both Ets1 and Fli1 occupy the CCN2 promoter. TGF-beta stimulation resulted in displacement of Fli1 from the CCN2 promoter and a transient inhibition of Fli1 synthesis. Moreover, reduction of Fli1 expression resulted in up-regulation of COL1A1 and COL1A2 genes and down-regulation of the MMP1 gene. Thus, inhibition of Fli1 recapitulated some of the key effects of TGF-beta, suggesting that Fli1 suppression is involved in activation of the profibrotic gene program in fibroblasts. On the other hand, activation of the CCN2 gene downstream of Ets1 is consistent with its role in angiogenesis and extracellular matrix remodeling. This study strongly supports a critical role of Fli1 and Ets1 in the pathological extracellular matrix regulation during fibrosis and cancer.

Modulation of transforming growth factor-beta (TGF-beta) signaling by endogenous sphingolipid mediators.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor that plays a critical role in tissue repair and fibrosis. Sphingolipid signaling has been shown to regulate a variety of cellular processes and has been implicated in collagen gene regulation. The present study was undertaken to determine whether endogenous sphingolipids are involved in the TGF-beta signaling pathway. TGF-beta treatment induced endogenous ceramide levels in a time-dependent manner within 5-15 min of cell stimulation. Using human fibroblasts transfected with a alpha2(I) collagen promoter/reporter gene construct (COL1A2), C(6)-ceramide (10 microm) exerted a stimulatory effect on basal and TGF-beta-induced activity of this promoter. Next, to define the effects of endogenous sphingolipids on TGF-beta signaling we employed ectopic expression of enzymes involved in sphingolipid metabolism. Sphingosine 1-phosphate phosphatase (YSR2) stimulated basal COL1A2 promoter activity and cooperated with TGF-beta in activation of this promoter. Furthermore, overexpression of YSR2 resulted in the pronounced increase of COL1A1 and COL1A2 mRNA levels. Conversely, overexpression of sphingosine kinase (SPHK1) inhibited basal and TGF-beta-stimulated COL1A2 promoter activity. These results suggest that endogenous ceramide, but not sphingosine or sphingosine 1-phosphate, is a positive regulator of collagen gene expression. Mechanistically, we demonstrate that Smad3 is a target of YSR2. TGF-beta-induced Smad3 phosphorylation was elevated in the presence of YSR2. Cotransfection of YSR2 with wild-type Smad3, but not with the phosphorylation-deficient mutant of Smad3 (Smad3A), resulted in a dramatic increase of COL1A2 promoter activity. In conclusion, this study demonstrates a direct role for the endogenous sphingolipid mediators in regulating the TGF-beta signaling pathway.

Persistent down-regulation of Fli1, a suppressor of collagen transcription, in fibrotic scleroderma skin.

The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo. This conclusion is supported by the analyses of mouse embryonic fibroblasts from Fli1(-/-), Fli1(+/-), and Fli1(+/+) mice. In cultured human and mouse fibroblasts Fli1 expression levels are inversely correlated with the collagen type I expression levels. These in vitro observations were validated in vivo. In healthy human skin Fli1 protein is expressed in fibroblasts and endothelial cells. Significantly, absence of Fli1 expression in individual fibroblasts correlates with elevated collagen synthesis. In contrast to healthy skin, Fli1 protein is consistently absent from fibroblasts and significantly reduced in endothelial cells in clinically involved scleroderma skin, which correlates with enhanced collagen synthesis in systemic sclerosis skin. This study supports the role of Fli1 as a suppressor of collagen transcription in human skin in vivo. Persistent down-regulation of Fli1 in scleroderma fibroblasts in vivo may directly contribute to uncontrolled matrix deposition in scleroderma skin.