Guadalupe Villarreal

Adaptive evolution of pelvic reduction in sticklebacks by recurrent deletion of a Pitx1 enhancer

Adaptive Girdle Loss in Sticklebacks How do molecular changes give rise to phenotypic adaptation exemplified by the repeated reduction in the pelvic girdle observed in separate populations of sticklebacks? Now Chan et al. (p. 302, published online 10 December) have identified the specific DNA changes that control this major skeletal adaptation. The key locus controlling pelvic phenotypes mapped to a noncoding regulatory region upstream of the Pituitary homeobox transcription factor 1 gene, which drives a tissue-specific pelvic enhancer. Multiple populations showed independent deletions in this region and enhancer function was inactivated. Reintroduction of the enhancer restored pelvic development in a pelvic-reduced stickleback. Loss of a tissue-specific enhancer explains multiple parallel losses of the pelvic girdle in stickleback populations. The molecular mechanisms underlying major phenotypic changes that have evolved repeatedly in nature are generally unknown. Pelvic loss in different natural populations of threespine stickleback fish has occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The high prevalence of deletion mutations at Pitx1 may be influenced by inherent structural features of the locus. Although Pitx1 null mutations are lethal in laboratory animals, Pitx1 regulatory mutations show molecular signatures of positive selection in pelvic-reduced populations. These studies illustrate how major expression and morphological changes can arise from single mutational leaps in natural populations, producing new adaptive alleles via recurrent regulatory alterations in a key developmental control gene.

Activation of SIRT1 by resveratrol induces KLF2 expression conferring an endothelial vasoprotective phenotype

AIMS Resveratrol activates Sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide-dependent deacetylase which modulates metabolic homeostasis and improves several pathophysiological features present in diseases of ageing. In particular, it has been shown that SIRT1 activation improves endothelial dysfunction and suppresses vascular inflammation, two central pathophysiological processes involved in the initiation and progression of cardiovascular disease. The downstream targets of SIRT1 activation in this context, however, remain poorly defined. Therefore, in this study, we aimed to characterize mechanistically how SIRT1 activation regulates the endothelial vasoprotective phenotype. METHODS AND RESULTS We demonstrate that SIRT1 activation by resveratrol increases the expression of the transcription factor Krüppel-like factor 2 (KLF2) in human vascular endothelial cells, resulting in the orchestrated regulation of transcriptional programs critical for conferring an endothelial vasoprotective phenotype. Moreover, we show that KLF2 upregulation by resveratrol occurs via a mitogen-activated protein kinase 5/myocyte enhancing factor 2-dependent signalling pathway. CONCLUSION Collectively, these observations provide a new mechanistic framework to understand the vascular protective effects mediated by SIRT1 activators and define KLF2 as a critical mediator of these effects.

Defining the regulation of KLF4 expression and its downstream transcriptional targets in vascular endothelial cells.

The Kruppel-like factor 2 (KLF2) and Kruppel-like factor 4 (KLF4) transcription factors have recently been shown to act as critical regulators of endothelial homeostasis. While several insights have been made into the signaling mechanisms orchestrating endothelial KLF2 expression, those governing the expression of KLF4 in the vascular endothelium remain largely unknown. Here, we show that diverse vasoprotective stimuli including an atheroprotective shear stress waveform, simvastatin, and resveratrol induce the expression of KLF4 in cultured human endothelial cells. We further demonstrate that the induction of KLF4 by resveratrol and atheroprotective shear stress occurs via a MEK5/MEF2-dependent signaling pathway. Since MEK5 activation is also critical for the expression of KLF2, we assessed the individual contribution of KLF4 and KLF2 to the global transcriptional activity triggered by MEK5 activation. Genome-wide transcriptional profiling of endothelial cells overexpressing KLF4, KLF2, or constitutively active MEK5 revealed that 59.2% of the genes regulated by the activation of MEK5 were similarly controlled by either KLF2 or KLF4. Collectively, our data identify a significant degree of mechanistic and functional conservation between KLF2 and KLF4, and importantly, provide further insights into the complex regulatory networks governing endothelial vasoprotection.

Flow cessation triggers endothelial dysfunction during organ cold storage conditions: strategies for pharmacologic intervention.

Background. Vascular pathologies constitute a major cause of graft rejection after organ transplantation. Recent studies have documented an improvement in transplant outcome when organs are preserved through pulsatile perfusion; however, the underlying mechanisms of these observations are poorly characterized. We hypothesized that the temporary absence of flow occurring in the context of organ cold storage conditions disrupts endothelial vasoprotective programs, and that this consequence of stasis may be a target for pharmacological modulation. Methods. The expression of the transcription factor Kruppel-like factor 2 (KLF2) and its vasoprotective target genes were assessed during cold storage conditions in cultured human endothelial cells and murine aortic segments. In addition, we evaluated the effect of simvastatin used as a supplement in a cold preservation solution on the expression of vasoprotective genes, and on endothelial activation and apoptosis. Results. The expression of endothelial KLF2 and its vasoprotective transcriptional targets were rapidly lost during cold preservation in vitro and ex vivo. Importantly, simvastatin treatment blocked the decay of KLF2, sustaining a vasoprotective phenotype, and preventing endothelial activation and apoptosis. Conclusions. Flow stasis leads to acute endothelial dysfunction and apoptosis in the context of cold storage conditions. Supplementation of organ preservation solutions with pharmacologic agents that restore endothelial vasoprotective programs, by upregulating KLF2, may represent a significant advancement of current organ preservation techniques.

Coordinated regulation of extracellular matrix synthesis by the microRNA-29 family in the trabecular meshwork.

PURPOSE The microRNA-29 (miR-29) family has emerged, in various tissues, as a key modulator of extracellular matrix (ECM) homeostasis. In this study, the authors investigate the role of the miR-29 family in the regulation of ECM synthesis in the trabecular meshwork (TM) under basal and TGF-β2 stimulatory conditions. METHODS Human TM cells were incubated with 2.5 ng/mL activated, recombinant human TGF-β2 for 24, 48, and 72 hours. A specific pharmacologic inhibitor was used to block SMAD3 function in the context of TGF-β2 stimulation. Changes in the expression of the miR-29 family were assessed by real-time PCR. The effect of miR-29 molecules and inhibitors on ECM levels was determined by immunoblot analysis. RESULTS All three members of the miR-29 family were expressed in cultured TM cells. Although the incubation of TM cells with TGF-β2 induced miR-29a and suppressed miR-29b levels, no significant effect was observed on miR-29c expression. Additional studies revealed that SMAD3 modulates miR-29b expression under basal and TGF-β2 conditions. Subsequent gain- and loss-of-function experiments demonstrated that the miR-29 family functions as a critical suppressor of various ECM proteins under basal and TGF-β2 stimulatory conditions. CONCLUSIONS The findings derived from this study identify the miR-29 family as a critical regulator of ECM expression in the TM and suggest that its modulation by TGF-β2 may be important in controlling ECM synthesis. Together, these data provide further insight into the complex regulatory mechanisms mediating TGF-β2 signaling and ECM production in the TM.

Identification of a Steap3 endosomal targeting motif essential for normal iron metabolism

Hereditary forms of iron-deficiency anemia, including animal models, have taught us much about the normal physiologic control of iron metabolism. However, the discovery of new informative mutants is limited by the natural mutation frequency. To address this limitation, we have developed a screen for heritable abnormalities of red blood cell morphology in mice with single-nucleotide changes induced by the chemical mutagen ethylnitrosourea (ENU). We now describe the first strain, fragile-red, with hypochromic microcytic anemia resulting from a Y228H substitution in the ferrireductase Steap3 (Steap3(Y288H)). Analysis of the Steap3(Y288H) mutant identifies a conserved motif required for targeting Steap3 to internal compartments and highlights how phenotypic screens linked to mutagenesis can identify new functional variants in erythropoiesis and ascribe function to previously unidentified motifs.

Canonical wnt signaling regulates extracellular matrix expression in the trabecular meshwork.

PURPOSE Canonical Wnt signaling has emerged as a critical regulator of aqueous outflow facility and intraocular pressure (IOP). In this study, we examine the role of canonical Wnt signaling on extracellular matrix (ECM) expression in the trabecular meshwork (TM) and explore the molecular mechanisms involved. METHODS β-catenin localization in human TM tissue was examined using immunofluorescent staining. Primary human TM cells were incubated with lithium chloride (LiCl) and the effect on active β-catenin expression was assessed by immunoblot. Adenovirus expressing a dominant-negative TCF4 mutant that lacks a β-catenin binding domain was used. Changes in the levels of the microRNA-29 (miR-29) family and ECM proteins were determined by real-time quantitative PCR and immunoblot analysis, respectively. RESULTS β-catenin was expressed throughout the TM, with localization primarily to the plasma membrane. Incubation of TM cells with lithium chloride increased the expression of active β-catenin. Lithium chloride treatment upregulated miR-29b expression, and suppressed the levels of various ECM proteins under both basal and TGF-β2 stimulatory conditions. Infection of TM cells with a dominant-negative TCF4 mutant induced ECM levels without a significant change in the expression of the miR-29 family. CONCLUSIONS Collectively, our data identify the canonical Wnt signaling pathway as an important modulator of ECM expression in the TM and provide a mechanistic framework for its regulation of outflow facility and IOP.

Pharmacological Regulation of SPARC by Lovastatin in Human Trabecular Meshwork Cells

PURPOSE Statins have been shown to increase aqueous outflow facility. The matricellular protein SPARC (secreted protein acidic and rich in cysteine) is a critical mediator of aqueous outflow and intraocular pressure (IOP). Here, we examine the effects of lovastatin on SPARC expression in trabecular meshwork (TM) cells, exploring the molecular mechanisms involved. METHODS Primary cultured human TM cells were incubated for 24, 48, and 72 hours with 10 μM lovastatin. In separate cultures, media was supplemented with either farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) for the duration of the 72-hour time point experiment. Trabecular meshwork cells were also pretreated for 24 hours with lovastatin followed by 24-hour stimulation with 3 ng/mL TGF-β2. Cell lysates and media were harvested and relative mRNA and protein level changes were determined. Krüppel-like factor 4 (KLF4) localization in normal human anterior segments was examined by immunofluorescence. Adenovirus expressing human KLF4 was used and relative changes in SPARC mRNA and protein levels were assessed. RESULTS Incubating TM cells with lovastatin suppressed SPARC mRNA and protein levels. This effect was reversed upon media supplementation with GGPP but not FPP. Pretreating cells with lovastatin inhibited TGF-β2 induction of SPARC. The KLF4 transcription factor was expressed throughout the TM and the inner and outer walls of Schlemms canal. Lovastatin treatment upregulated KLF4 mRNA and protein levels. Overexpression of KLF4 downregulated SPARC expression. CONCLUSIONS Collectively, our data identify lovastatin as an important pharmacological suppressor of SPARC expression in TM cells, and provide further insight into the molecular mechanisms mediating statin enhancement of aqueous outflow facility.

AMP-activated protein kinase regulates intraocular pressure, extracellular matrix, and cytoskeleton in trabecular meshwork

PURPOSE In this study, we investigate how adenosine monophosphate-activated protein kinase (AMPK) affects extracellular matrix (ECM) and cellular tone in the trabecular meshwork (TM), and examine how deletion of its catalytic α2 subunit affects IOP and aqueous humor clearance in mice. METHODS Human TM tissue was examined for expression of AMPKα1 and AMPKα2, genomically distinct isoforms of the AMPK catalytic subunit. Primary cultured human TM cells were treated for 24 hours with the AMPK activator 5-amino-1-β-Dffff-ribofuranosyl-imidazole-4-carboxamide (AICAR), under basal or TGF-β2 stimulatory conditions. Conditioned media (CM) was probed for secreted protein acidic and rich in cysteine (SPARC), thrombospondin-1 (TSP-1), and ECM proteins, and cells were stained for F-actin. Cells underwent adenoviral infection with a dominant negative AMPKα subunit (ad.DN.AMPKα) and were similarly analyzed. Intraocular pressure, central corneal thickness (CCT), and aqueous clearance were measured in AMPKα2-null and wild-type (WT) mice. RESULTS Both AMPKα1 and AMPKα2 are expressed in TM. AICAR activated AMPKα and suppressed the expression of various ECM proteins under basal and TGF-β2 stimulatory conditions. AICAR decreased F-actin staining and increased the phospho-total RhoA ratio (Ser188). Transforming growth factor-β2 transiently dephosphorylated AMPKα. Infection with ad.DN.AMPKα upregulated various ECM proteins, decreased the phospho-total RhoA ratio, and increased F-actin staining. AMPKα2-null mice exhibited 6% higher IOP and decreased aqueous clearance compared with WT mice, without significant differences in CCT or angle morphology. CONCLUSIONS Collectively, our data identify AMPK as a critical regulator of ECM homeostasis and cytoskeletal arrangement in the TM. Mice that are AMPKα2-null exhibit higher IOPs and decreased aqueous clearance than their WT counterparts.

Krüppel-like Factors in the Vascular Endothelium

Although Kruppel-like factors (KLFs) have been the subject of extensive biological investigation, the role of this family of transcription factors in the biology and pathophysiology of the vascular endothelium is just becoming apparent. Most investigative efforts thus far have focused on KLF2, its contribution to the endothelial vasoprotective phenotype, and its possible impact on atherogenesis. This chapter reflects on the current state of the field and highlights evolving areas where KLFs are emerging as important regulators of endothelial function.