Purinergic dysregulation in pulmonary hypertension.

Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.

Genomic copy number analysis of a spectrum of blue nevi identifies recurrent aberrations of entire chromosomal arms in melanoma ex blue nevus.

Blue nevi may display significant atypia or undergo malignant transformation. Morphologic diagnosis of this spectrum of lesions is notoriously difficult, and molecular tools are increasingly used to improve diagnostic accuracy. We studied copy number aberrations in a cohort of cellular blue nevi, atypical cellular blue nevi, and melanomas ex blue nevi using Affymetrix's OncoScan platform. Cases with sufficient DNA were analyzed for GNAQ, GNA11, and HRAS mutations. Copy number aberrations were detected in 0 of 5 (0%) cellular blue nevi, 3 of 12 (25%) atypical cellular blue nevi, and 6 of 9 (67%) melanomas ex blue nevi. None of the atypical cellular blue nevi displayed more than one aberration, whereas complex aberrations involving four or more regions were seen exclusively in melanomas ex blue nevi. Gains and losses of entire chromosomal arms were identified in four of five melanomas ex blue nevi with copy number aberrations. In particular, gains of 1q, 4p, 6p, and 8q, and losses of 1p and 4q were each found in at least two melanomas. Whole chromosome aberrations were also common, and represented the sole finding in one atypical cellular blue nevus. When seen in melanomas, however, whole chromosome aberrations were invariably accompanied by partial aberrations of other chromosomes. Three melanomas ex blue nevi harbored aberrations, which were absent or negligible in their precursor components, suggesting progression in tumor biology. Gene mutations involving GNAQ and GNA11 were each detected in two of eight melanomas ex blue nevi. In conclusion, copy number aberrations are more common and often complex in melanomas ex blue nevi compared with cellular and atypical cellular blue nevi. Identification of recurrent gains and losses of entire chromosomal arms in melanomas ex blue nevi suggests that development of new probes targeting these regions may improve detection and risk stratification of these lesions.

Elevated YAP and its downstream targets CCN1 and CCN2 in basal cell carcinoma: impact on keratinocyte proliferation and stromal cell activation.

Yes-associated protein (YAP) is a transcriptional co-activator of hippo signaling pathway, which plays an important role in organ size control and tumorigenesis. Here we report that YAP and its downstream transcriptional targets CCN1 and CCN2 are markedly elevated in keratinocytes in human skin basal cell carcinoma tumor islands. In human keratinocytes, knockdown of YAP significantly reduced expression of CCN1 and CCN2, and repressed proliferation and survival. This inhibition of proliferation and survival was rescued by restoration of CCN1 expression, but not by CCN2 expression. In basal cell carcinoma stroma, CCN2-regulated genes type I collagen, fibronectin, and α-smooth muscle actin were highly expressed. Furthermore, atomic force microscopy revealed increased tissue stiffness in basal cell carcinoma stroma compared to normal dermis. These data provide evidence that up-regulation of YAP in basal cell carcinoma impacts both aberrant keratinocyte proliferation, via CCN1, and tumor stroma cell activation and stroma remodeling, via CCN2. Targeting YAP and/or CCN1 and CCN2 may provide clinical benefit in basal cell carcinoma.

Alterations of Dermal Connective Tissue Collagen in Diabetes: Molecular Basis of Aged-Appearing Skin.

Alterations of the collagen, the major structural protein in skin, contribute significantly to human skin connective tissue aging. As aged-appearing skin is more common in diabetes, here we investigated the molecular basis of aged-appearing skin in diabetes. Among all known human matrix metalloproteinases (MMPs), diabetic skin shows elevated levels of MMP-1 and MMP-2. Laser capture microdissection (LCM) coupled real-time PCR indicated that elevated MMPs in diabetic skin were primarily expressed in the dermis. Furthermore, diabetic skin shows increased lysyl oxidase (LOX) expression and higher cross-linked collagens. Atomic force microscopy (AFM) further indicated that collagen fibrils were fragmented/disorganized, and key mechanical properties of traction force and tensile strength were increased in diabetic skin, compared to intact/well-organized collagen fibrils in non-diabetic skin. In in vitro tissue culture system, multiple MMPs including MMP-1 and MM-2 were induced by high glucose (25 mM) exposure to isolated primary human skin dermal fibroblasts, the major cells responsible for collagen homeostasis in skin. The elevation of MMPs and LOX over the years is thought to result in the accumulation of fragmented and cross-linked collagen, and thus impairs dermal collagen structural integrity and mechanical properties in diabetes. Our data partially explain why old-looking skin is more common in diabetic patients.

Oxidant exposure induces cysteine-rich protein 61 (CCN1) via c-Jun/AP-1 to reduce collagen expression in human dermal fibroblasts.

Human skin is a primary target of oxidative stress from reactive oxygen species (ROS) generated from both extrinsic and intrinsic sources. Oxidative stress inhibits the production of collagen, the most abundant protein in skin, and thus contributes to connective tissue aging. Here we report that cysteine-rich protein 61 (CCN1), a negative regulator of collagen production, is markedly induced by ROS and mediates loss of type I collagen in human dermal fibroblasts. Conversely, antioxidant N-acetyl-L-cysteine significantly reduced CCN1 expression and prevented ROS-induced loss of type I collagen in both human dermal fibroblasts and human skin in vivo. ROS increased c-Jun, a critical member of transcription factor AP-1 complex, and increased c-Jun binding to the AP-1 site of the CCN1 promoter. Functional blocking of c-Jun significantly reduced CCN1 promoter and gene expression and thus prevented ROS-induced loss of type I collagen. Targeting the c-Jun/CCN1 axis may provide clinical benefit for connective tissue aging in human skin.

Robust shifts in S100a9 expression with aging: a novel mechanism for chronic inflammation.

The S100a8 and S100a9 genes encode a pro-inflammatory protein (calgranulin) that has been implicated in multiple diseases. However, involvement of S100a8/a9 in the basic mechanisms of intrinsic aging has not been established. In this study, we show that shifts in the abundance of S100a8 and S100a9 mRNA are a robust feature of aging in mammalian tissues, involving a range of cell types including the central nervous system. To identify transcription factors that control S100a9 expression, we performed a large-scale transcriptome analysis of 62 mouse and human cell types. We identified cell type-specific trends, as well as robust associations linking S100a9 coexpression to elevated frequency of ETS family motifs, and in particular, to motifs recognized by the transcription factor SPI/PU.1. Sparse occurrence of SATB1 motifs was also a strong predictor of S100a9 coexpression. These findings offer support for a novel mechanism by which a SPI1/PU.1-S100a9 axis sustains chronic inflammation during aging.

CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts.

Dermal connective tissue collagen is the major structural protein in skin. Fibroblasts within the dermis are largely responsible for collagen production and turnover. We have previously reported that dermal fibroblasts, in aged human skin in vivo, express elevated levels of CCN1, and that CCN1 negatively regulates collagen homeostasis by suppressing collagen synthesis and increasing collagen degradation (Quan et al. Am J Pathol 169:482-90, 2006, J Invest Dermatol 130:1697-706, 2010). In further investigations of CCN1 actions, we find that CCN1 alters collagen homeostasis by promoting expression of specific secreted proteins, which include matrix metalloproteinases and proinflammatory cytokines. We also find that CCN1-induced secretory proteins are elevated in aged human skin in vivo. We propose that CCN1 induces an "Age-Associated Secretory Phenotype", in dermal fibroblasts, which mediates collagen reduction and fragmentation in aged human skin.