Cell competition and fitness comparison between cancer and tumor microenvironment (TME) cells determine oncogenic fate. Our previous study established a role for human Flower isoforms as fitness fingerprints, where the expression of Flower Win isoforms in tumor cells leads to growth advantage over TME cells expressing Lose isoforms. Here we demonstrate that the expression of Flower Lose and reduced microenvironment fitness is not a pre-existing condition but, rather, a cancer-induced phenomenon. Cancer cells actively reduce TME fitness by the exosome-mediated release of a cancer-specific long non-coding RNA, Tu-Stroma, which controls the splicing of the Flower gene in the TME cells and expression of Flower Lose isoform, which leads to reduced fitness status. This mechanism controls cancer growth, metastasis and host survival in ovarian cancer. Targeting Flower protein with humanized monoclonal antibody (mAb) in mice significantly reduces cancer growth and metastasis and improves survival. Pre-treatment with Flower mAb protects intraperitoneal organs from developing lesions despite the presence of aggressive tumor cells.

Epigenetic programming of gene expression is vital to normal physiology, and dysregulation of epigenetic proteins correlates with numerous diseases. Gene expression is known to be regulated through DNA and histone modifications. However, it is complicated to assess systematic changes caused by epigenetic modulators. Tools for epigenetic drug discovery continue to be developed through structure-activity relationship studies. Phenotypic profiling of human primary cells and cell lines opens the gateway to target identification and lead optimization to guide drug discovery campaigns. Epigenetic proteins, including readers, writers, and erasers, are putative drug targets in many diseases. Profiling assays provide insight into molecular mechanisms of epigenetic targets, predictive biomarker analysis, and chemical safety assessment. In this chapter, we highlight the advancements in epigenetic drug development via structure-activity relationship studies and cellular/phenotypic profiling assays. More extensive profiling system databases and focused chemical libraries will aid medicinal chemistry efforts toward development of small molecule epigenetic modulators that can have high implications in disease treatment.

Risk stratification of COVID-19 patients is essential for pandemic management. Changes in the cell fitness marker, hFwe-Lose, can precede the host immune response to infection, potentially making such a biomarker an earlier triage tool. Here, we evaluate whether hFwe-Lose gene expression can outperform conventional methods in predicting outcomes (e.g., death and hospitalization) in COVID-19 patients. We performed a post-mortem examination of infected lung tissue in deceased COVID-19 patients to determine hFwe-Lose's biological role in acute lung injury. We then performed an observational study (n = 283) to evaluate whether hFwe-Lose expression (in nasopharyngeal samples) could accurately predict hospitalization or death in COVID-19 patients. In COVID-19 patients with acute lung injury, hFwe-Lose is highly expressed in the lower respiratory tract and is co-localized to areas of cell death. In patients presenting in the early phase of COVID-19 illness, hFwe-Lose expression accurately predicts subsequent hospitalization or death with positive predictive values of 87.8-100% and a negative predictive value of 64.1-93.2%. hFwe-Lose outperforms conventional inflammatory biomarkers and patient age and comorbidities, with an area under the receiver operating characteristic curve (AUROC) 0.93-0.97 in predicting hospitalization/death. Specifically, this is significantly higher than the prognostic value of combining biomarkers (serum ferritin, D-dimer, C-reactive protein, and neutrophil-lymphocyte ratio), patient age and comorbidities (AUROC of 0.67-0.92). The cell fitness marker, hFwe-Lose, accurately predicts outcomes in COVID-19 patients. This finding demonstrates how tissue fitness pathways dictate the response to infection and disease and their utility in managing the current COVID-19 pandemic.

Cancer is a complex disease with high incidence and mortality rates. The important role played by the tumor microenvironment in regulating oncogenesis, tumor growth, and metastasis is by now well accepted in the scientific community. SPARC is known to participate in tumor-stromal interactions and impact cancer growth in ambiguous ways, which either enhance or suppress cancer aggressiveness, in a context-dependent manner. p53 transcription factor, a well-established tumor suppressor, has been reported to promote tumor growth in certain situations, such as hypoxia, thus displaying a duality in its action. Although both proteins are being tested in clinical trials, the synergistic relation between them is yet to be explored in clinical practice. In this review, we address the controversial roles of SPARC and p53 as double agents in cancer, briefly summarizing the interaction found between these two molecules and its importance in cancer.

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.

In many cases, cancers are difficult to eliminate because they develop resistance to a primary chemotherapy or targeted therapy. Tumors grow into diverse cell subpopulations, increasing the ability to resist elimination. The phenomenon of ‘cell competition’ describes our body’s natural surveillance system to optimize tissue fitness by forcing viable but aberrant cells to undergo cell death. Cell competition is not simply comparison of cell division potential. Competition factors signal for ‘loser’ cell elimination and ‘winner’ cell dominance. New evidence demonstrates it is possible to restrict cancer growth by strengthening the cell fitness of surrounding healthy tissue via anti-apoptotic pathways. Hence, cell competition provides strong conceptual explanation for oncogenesis, tumor growth and suppression. Tumor heterogeneity is a hallmark of many cancers and establishes gradients in which competitive interactions are able to occur among tumor cell subpopulations as well as neighboring stromal tissue. Here we review cellular/molecular competition pathways in the context of tumor evolution, heterogeneity and response to interventions. We propose strategies to exploit these mediators and design novel broad-spectrum therapeutic approaches that eliminate cancer and enhance fitness of neighboring tissue to improve patient outcomes.

In humans, the adaptive immune system uses the exchange of information between cells to detect and eliminate foreign or damaged cells; however, the removal of unwanted cells does not always require an adaptive immune system(1,2). For example, cell selection in Drosophila uses a cell selection mechanism based on 'fitness fingerprints', which allow it to delay ageing(3), prevent developmental malformations(3,4) and replace old tissues during regeneration(5). At the molecular level, these fitness fingerprints consist of combinations of Flower membrane proteins(3,4,6). Proteins that indicate reduced fitness are called Flower-Lose, because they are expressed in cells marked to be eliminated(6). However, the presence of Flower-Lose isoforms at a cell's membrane does not always lead to elimination, because if neighbouring cells have similar levels of Lose proteins, the cell will not be killed(4,6,7). Humans could benefit from the capability to recognize unfit cells, because accumulation of damaged but viable cells during development and ageing causes organ dysfunction and disease(8-17). However, in Drosophila this mechanism is hijacked by premalignant cells to gain a competitive growth advantage(18). This would be undesirable for humans because it might make tumours more aggressive(19-21). It is unknown whether a similar mechanism of cell-fitness comparison is present in humans. Here we show that two human Flower isoforms (hFWE1 and hFWE3) behave as Flower-Lose proteins, whereas the other two isoforms (hFWE2 and hFWE4) behave as Flower-Win proteins. The latter give cells a competitive advantage over cells expressing Lose isoforms, but Lose-expressing cells are not eliminated if their neighbours express similar levels of Lose isoforms; these proteins therefore act as fitness fingerprints. Moreover, human cancer cells show increased Win isoform expression and proliferate in the presence of Lose-expressing stroma, which confers a competitive growth advantage on the cancer cells. Inhibition of the expression of Flower proteins reduces tumour growth and metastasis, and induces sensitivity to chemotherapy. Our results show that ancient mechanisms of cell recognition and selection are active in humans and affect oncogenic growth.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

Preeclampsia is a cardiovascular disorder of late pregnancy that is, commonly characterized by hypertension, renal structural damage and dysfunction, and fetal growth restriction. Prevailing etiologic models of this disorder include T-cell dysfunction as an initiating cause of preeclampsia. Indoleamine 2,3-dioxygenase (IDO), an enzyme that mediates the conversion of tryptophan to kynurenine, has been linked to preeclampsia in humans, and is known to regulate T-cell activity and an endothelial-derived relaxing factor. To test the hypothesis that IDO is causally involved in the pathogenesis of preeclampsia, mice deficient for IDO (IDO-KO) were generated on a C57BL/6 background. IDO-KO and wild-type C57BL/6 mice were bred, and preeclampsia phenotypes were evaluated during pregnancy. Pregnant IDO-KO mice exhibited pathognomonic renal glomerular endotheliosis, proteinuria, pregnancy-specific endothelial dysfunction, intrauterine growth restriction, and mildly elevated blood pressure compared to wild-type mice. Together these findings highlight an important role for IDO in the generation of phenotypes typical of preeclampsia. Loss of IDO function may represent a risk factor for the development of preeclampsia. By extension, increased IDO activity, reductions in IDO reactants, or increases in IDO products may represent novel therapeutic approaches for this disorder.

Although peroxisome proliferator–activated receptor-γ (PPARγ) is thought to play a protective role in the vasculature, its cell-specific effect, particularly in resistance vessels, is poorly defined. Nitric oxide (NO) plays a major role in vascular biology in the brain. We examined the hypothesis that selective interference with PPARγ in vascular muscle would impair NO-dependent responses and augment vasoconstrictor responses in the cerebral circulation. We studied mice expressing a dominant negative mutation in human PPARγ (P467L) under the control of the smooth muscle myosin heavy chain promoter (S-P467L). In S-P467L mice, dilator responses to exogenously applied or endogenously produced NO were greatly impaired in cerebral arteries in vitro and in small cerebral arterioles in vivo. Select NO-independent responses, including vasodilation to low concentrations of potassium, were also impaired in S-P467L mice. In contrast, increased expression of wild-type PPARγ in smooth muscle had little effect on vasomotor responses. Mechanisms underlying impairment of both NO-dependent and NO-independent vasodilator responses after interference with PPARγ involved Rho kinase with no apparent contribution by oxidative stress–related mechanisms. These findings support the concept that via effects on Rho kinase–dependent signaling, PPARγ in vascular muscle is a major determinant of vascular tone in resistance vessels and, in particular, NO-mediated signaling in cerebral arteries and brain microvessels. Considering the importance of NO and Rho kinase, these findings have implications for regulation of cerebral blood flow and the pathogenesis of large and small vessel disease in brain.

Myogenic responses by resistance vessels are a key component of autoregulation in brain, thus playing a crucial role in regulating cerebral blood flow and protecting the blood–brain barrier against potentially detrimental elevations in blood pressure. Although cerebrovascular disease is often accompanied by alterations in myogenic responses, mechanisms that control these changes are poorly understood. Peroxisome proliferator–activated receptor γ has emerged as a regulator of vascular tone. We hypothesized that interference with peroxisome proliferator–activated receptor γ in smooth muscle would augment myogenic responses in cerebral arteries. We studied transgenic mice expressing a dominant-negative mutation in peroxisome proliferator–activated receptor γ selectively in smooth muscle (S-P467L) and nontransgenic littermates. Myogenic tone in middle cerebral arteries from S-P467L was elevated 3-fold when compared with nontransgenic littermates. Rho kinase is thought to play a major role in cerebrovascular disease. The Rho kinase inhibitor, Y-27632, abolished augmented myogenic tone in middle cerebral arteries from S-P467L mice. CN-03, which modifies RhoA making it constitutively active, elevated myogenic tone to ≈60% in both strains, via a Y-27632–dependent mechanism. Large conductance Ca 2+ -activated K + channels (BK Ca ) modulate myogenic tone. Inhibitors of BK Ca caused greater constriction in middle cerebral arteries from nontransgenic littermates when compared with S-P467L. Expression of RhoA or Rho kinase-I/II protein was similar in cerebral arteries from S-P467L mice. Overall, the data suggest that peroxisome proliferator–activated receptor γ in smooth muscle normally inhibits Rho kinase and promotes BK Ca function, thus influencing myogenic tone in resistance arteries in brain. These findings have implications for mechanisms that underlie large- and small-vessel disease in brain, as well as regulation of cerebral blood flow.

Hereditary hemochromatosis (HH) is a common autosomal recessive disorder of iron overload among Caucasians of northern European descent. Over 85% of all cases with HH are due to mutations in the hemochromatosis protein (HFE) involved in iron metabolism. Although the importance in iron homeostasis is well recognized, the mechanism of sensing and regulating iron absorption by HFE, especially in the absence of iron response element in its gene, is not fully understood. In this report, we have identified an inverted repeat sequence (ATGGTcttACCTA) within 1700bp (-1675/+35) of the HFE promoter capable to form cruciform structure that binds PARP1 and strongly represses HFE promoter. Knockdown of PARP1 increases HFE mRNA and protein. Similarly, hemin or FeCl3 treatments resulted in increase in HFE expression by reducing nuclear PARP1 pool via its apoptosis induced cleavage, leading to upregulation of the iron regulatory hormone hepcidin mRNA. Thus, PARP1 binding to the inverted repeat sequence on the HFE promoter may serve as a novel iron sensing mechanism as increased iron level can trigger PARP1 cleavage and relief of HFE transcriptional repression.

Agonists of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) have potent insulin-sensitizing effects and inhibit atherosclerosis progression in patients with Type II diabetes. Conversely, missense mutations in the ligand-binding domain of PPARγ that render the transcription factor dominant negative (DN) cause early-onset hypertension and Type II diabetes. We tested the hypothesis that DN PPARγ-mediated interference of endogenous wild-type PPARγ in the endothelium and vascular smooth muscle exacerbates atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. Endothelium-specific expression of DN PPARγ on the ApoE(-/-) background unmasked significant impairment of endothelium-dependent relaxation in aortic rings, increased systolic blood pressure, altered expression of atherogenic markers (e.g., Cd36, Mcp1, Catalase), and enhanced diet-induced atherosclerotic lesion formation in aorta. Smooth muscle-specific expression of DN PPARγ, which induces aortic dysfunction and increased systolic blood pressure at baseline, also resulted in enhanced diet-induced atherosclerotic lesion formation in aorta on the ApoE(-/-) background that was associated with altered expression of a shared, yet distinct, set of atherogenic markers (e.g., Cd36, Mcp1, Osteopontin, Vcam1). In particular, induction of Osteopontin expression by smooth muscle-specific DN PPARγ correlated with increased plaque calcification. These data demonstrate that inhibition of PPARγ function specifically in the vascular endothelium or smooth muscle may contribute to cardiovascular disease.

An indispensable role for the brain renin-angiotensin system (RAS) has been documented in most experimental animal models of hypertension. To identify the specific efferent pathway activated by the brain RAS that mediates hypertension, we examined the hypothesis that elevated arginine vasopressin (AVP) release is necessary for hypertension in a double-transgenic model of brain-specific RAS hyperactivity (the "sRA" mouse model). sRA mice experience elevated brain RAS activity due to human angiotensinogen expression plus neuron-specific human renin expression. Total daily loss of the 4-kDa AVP prosegment (copeptin) into urine was grossly elevated (≥8-fold). Immunohistochemical staining for AVP was increased in the supraoptic nucleus of sRA mice (~2-fold), but no quantitative difference in the paraventricular nucleus was observed. Chronic subcutaneous infusion of a nonselective AVP receptor antagonist conivaptan (YM-087, Vaprisol, 22 ng/h) or the V(2)-selective antagonist tolvaptan (OPC-41061, 22 ng/h) resulted in normalization of the baseline (~15 mmHg) hypertension in sRA mice. Abdominal aortas and second-order mesenteric arteries displayed AVP-specific desensitization, with minor or no changes in responses to phenylephrine and endothelin-1. Mesenteric arteries exhibited substantial reductions in V(1A) receptor mRNA, but no significant changes in V(2) receptor expression in kidney were observed. Chronic tolvaptan infusion also normalized the (5 mmol/l) hyponatremia of sRA mice. Together, these data support a major role for vasopressin in the hypertension of mice with brain-specific hyperactivity of the RAS and suggest a primary role of V(2) receptors.

Myogenic responses by resistance vessels in brain play a crucial role in regulating cerebral blood flow and protecting the brain parenchyma and blood-brain barrier against potentially detrimental elevations in blood pressure. PPARγ is a transcription factor that may protect the vasculature by suppressing increases in vascular tone. Using transgenic mice expressing a dominant negative mutation of human PPARγ only in smooth muscle cells (S-P467L; TG), we have shown that augmented myogenic responses in mesenteric arteries are mediated by a mechanism that involved protein kinase C, but not ROCK. As ROCK is thought to play a major role in cerebrovascular disease, we hypothesized that interference with PPARγ in smooth muscle would augment myogenic responses in cerebral arteries via a ROCK dependent mechanism. We studied S-P467L TG mice and non-transgenic littermates (NT). Middle cerebral arteries (MCA) from TG mice generated more myogenic tone compared with NT (75 mmHg: 32±2% vs. 13±2% of max diameter). Myogenic reactivity was similarly augmented in MCA from TG mice from 15-150 mmHg whereas vessel diameter in Ca2+ free conditions was similar between groups. We used two approaches to evaluate the impact of ROCK in this model. First, the ROCK inhibitor, Y27632 (3 μM), abolished the augmented myogenic tone in MCA from TG mice (34±5% vs. 9±4% of max diameter). Second, treatment with a cell permeable molecule that modifies RhoA to make it constitutively active (CN-03) elevated myogenic tone to ~60% in both groups, which was reduced to ~10% by Y27632. The large conductance potassium (BK) channel modulates myogenic tone. The BK channel inhibitor, iberiotoxin, caused greater constriction in MCA from NT compared with TG (100 nM: NT, 14±2% vs. TG, 8±1% Δ tone at 75 mmHg). Although angiotensin II can activate ROCK, the AT1 receptor antagonist (losartan) did not affect myogenic tone in MCA from TG mice. Gene expression analysis revealed no change in mRNA for RhoA or ROCK1/2 expression in cerebral arteries from TG mice. These findings support the concept that in cerebral arteries, activity of ROCK has substantial effects on myogenic tone. The data also suggest that under normal conditions, PPARγ in smooth muscle inhibits ROCK, influencing myogenic tone in resistance arteries in brain.

Agonists of the nuclear hormone receptor PPARγ inhibit atherosclerosis progression; however, direct actions of PPARγ in the vessel wall during atherosclerosis remain undefined. We reported that transgenic mice expressing dominant negative (DN) PPARγ targeted to the vascular endothelium (E-DN) or smooth muscle (S-DN) on the apolipoprotein E-deficient (ApoE -/- ) background exhibit increased diet-induced atherosclerotic lesion area in aorta compared to non-transgenic (NT) control mice without changes in plasma cholesterol or triglycerides. Here we show that abdominal aortic rings from standard diet-fed E-DN.ApoE -/- mice display impaired endothelium-dependent relaxation in response to Ach (3μM: 44±2% vs 72±1% in NT.ApoE -/- ; P<0.05) despite normal relaxation to SNP (1μM: 85±1% vs 85±2%; NS) and contraction in response to ET-1 (30nM: 0.84±0.13g vs 0.75±0.08g; NS) and Ang II (10nM: 0.33±0.03g vs 0.33±0.04g; NS). In contrast, abdominal aortic rings from S-DN.ApoE -/- mice display impaired nitric oxide-dependent relaxation in response to Ach (3μM: 47±3% vs 71±1% in NT.ApoE -/- ; P<0.05) and SNP (1μM: 57±3% vs 84±2%; P<0.05) plus enhanced contraction to ET-1 (30nM: 0.98±0.05g vs 0.71±0.12g; P<0.05) and Ang II (10nM: 0.49±0.06g vs 0.36±0.03g; P<0.05). Systolic blood pressure was elevated in E-DN.ApoE -/- mice (127±3 vs 112±2 mmHg in NT.ApoE -/- ; P<0.05) and S-DN.ApoE -/- mice (123±1 vs 109±2 mmHg in NT.ApoE -/- ; P<0.05). Analyzed by real-time qPCR, aorta from E-DN.ApoE -/- mice had increased mRNA expression of Mcp1 (1.68±0.26-fold of NT.ApoE -/- ; P<0.05) and decreased expression of Catalase (0.59±0.13-fold; P<0.05) and Cd36 (0.69±0.09-fold; P<0.05), while Cd68 , Icam1 , Tnfα , Osteopontin , and Vcam1 were not changed. Aorta from S-DN.ApoE -/- mice had increased mRNA expression of Mcp1 (1.75±0.23-fold of NT.ApoE -/- ; P<0.05), Osteopontin (2.18±0.13-fold; P<0.05) and Vcam1 (1.87±0.24-fold; P<0.05) and decreased expression of Cd36 (0.72±0.08-fold; P<0.05), while Cd68 , Icam1 , Tnfα , and Catalase were not changed. In summary, endothelial- or smooth muscle-specific DN PPARγ induces distinct alterations in vascular function and atherogenic markers. Inhibition of PPARγ function in the vessel wall may contribute to cardiovascular disease.

Hypertension in many animal models is sensitive to inhibition of the brain renin-angiotensin system (RAS). We examined mice with transgenic hyperactivity of the brain RAS (sRA mice) to determine whether this manipulation is sufficient to cause hypertension, and to identify the causative mechanism. sRA mice exhibit brain-specific increases in angiotensin (ANG) peptide production through neuron-specific expression of human renin (synapsin promoter) and expression of human angiotensinogen through its own promoter. We determined both through tail-cuff and radiotelemetric methods that sRA mice are hypertensive (SBP; control 112±2 vs sRA 127±5 mmHg, P=0.02). Despite normal plasma osmolality and moderate hyponatremia, sRA mice exhibit double the number of vasopressin-expressing neurons in the supraoptic nucleus (P=0.002), as detected by immunohistochemistry. Plasma levels of the vasopressin pro-segment, copeptin, were reduced in sRA mice (140±19 vs 68±21 pg/mL, P=0.02), which correlated with severe polyuria (1.8±0.3 vs 12.3±1.6 mL/day, P<0.001). Indeed, total daily copeptin loss in the urine was significantly increased almost twenty-fold in sRA mice (7.9±4.3 vs 154.4±62.4 pg/day, P=0.03), highlighting an increase in vasopressin secretion per unit time. The baseline hypertension of sRA mice was completely reversed by chronic infusion of the dual V 1A / V 2 receptor antagonist, conivaptan (22 ng/hr, 10 days, s.c.; 113±5 mmHg, P=0.02). Preliminary experiments demonstrate that infusion of the selective V 2 receptor antagonist, tolvaptan (22 ng/hr, 10 days, s.c.), has similar effects. Further, while abdominal aorta and mesenteric arteries demonstrate selective desensitization to vasopressin and down-regulation of the V 1A receptor (38% of control, P<0.05), renal V 2 receptor expression remained normal in sRA mice. Together, these data demonstrate major roles for vasopressin and its V 2 receptor in the hypertension caused by elevated brain RAS activity.

Mutations in Cullin-3 result in early-onset hypertension in humans through an undefined mechanism. Cullin-3 RING E3 ubiquitin ligase complex mediates proteasomal degradation of RhoA, a key mediator of vascular smooth muscle tone. We reported that smooth muscle-specific expression of dominant negative PPARγ (S-P467L) in transgenic mice causes hypertension and aortic dysfunction via increased RhoA/Rho-kinase signaling. Dominant negative PPARγ caused decreased Cullin-3 protein (0.31±0.04-fold of Non-Transgenic (NT); P<0.05) and decreased ratio of active Nedd8-Cullin-3 / Cullin-3 (0.06±0.04 vs 0.51±0.18 NT; P<0.05), which correlated with increased protein levels of Cullin-3 substrates RhoA (2.6±0.2-fold of NT; P<0.05) and Cyclin E (2.0±0.1-fold of NT; P<0.05) in S-P467L medial aorta. We tested the hypothesis that Cullin-3 controls arterial pressure by regulating vascular function. siRNA-mediated knockdown of Cullin-3 (72 hr), confirmed by Western blot, increased RhoA (2.5±0.1-fold of NC1 negative control siRNA; P<0.05) and Cyclin E (1.9±0.2-fold of NC1; P<0.05) protein in primary rat aortic smooth muscle cells. Inhibition of cullin-RING ligase activity using the Nedd8-activating enzyme inhibitor, MLN4924 (1μM, 16 hr), also increased RhoA (4.3±1.0-fold of DMSO vehicle). Treatment of aortic rings from control mice with MLN4924 (1μM in DMEM/F12, 16 hr) resulted in enhanced agonist-mediated contraction in response to endothelin 1 (30nM: 0.35±0.05g vs 0.19±0.02g DMSO vehicle; P<0.05), serotonin (3μM: 1.2±0.07g vs 0.9±0.08g; P<0.05), and phenylephrine (3μM: 1.1±0.05g vs 0.7±0.05g, P<0.05) that was Rho-kinase-dependent, despite decreased contraction to KCl (100mM: 0.7±0.03g vs 1.0±0.03g; P<0.05). Finally, administration of MLN4924 (30mg/kg TID, 2 days) to control mice in vivo increased mean arterial pressure during the light phase (121±4 vs. 108±3mmHg baseline, P<0.05; dark phase: 122±4 vs. 123±4mmHg baseline, NS) as measured by radiotelemetry. Our results demonstrate that interference with PPARγ in smooth muscle causes vascular dysfunction via impaired Cullin-3-mediated regulation of RhoA/Rho-kinase signaling and provide a mechanistic link between mutations in Cullin-3 and hypertension.

Rationale: Activation of peroxisome proliferator−activated receptor-γ (PPARγ) by thiazolidinediones lowers blood pressure, whereas PPARγ mutations cause hypertension. Previous studies suggest these effects may be mediated through the vasculature, but the underlying mechanisms remain unclear. Objective: To identify PPARγ mechanisms and transcriptional targets in vascular smooth muscle and their role in regulating resistance artery tone. Methods and Results: We studied mesenteric artery (MA) from transgenic mice expressing dominant-negative (DN) mutant PPARγ driven by a smooth muscle cell−specific promoter. MA from transgenic mice exhibited a robust increase in myogenic tone. Patch clamp analysis revealed a reduced large conductance Ca 2+ -activated K + (BKCa) current in freshly dissociated smooth muscle cell from transgenic MA. Inhibition of protein kinase C corrected both enhanced myogenic constriction and impaired the large conductance Ca 2+ -activated K + channel function. Gene expression profiling revealed a marked loss of the regulator of G protein signaling 5 (RGS5) mRNA in transgenic MA, which was accompanied by a substantial increase in angiotensin II–induced constriction in MA. Small interfering RNA targeting RGS5 caused augmented myogenic tone in intact mesenteric arteries and increased activation of protein kinase C in smooth muscle cell cultures. PPARγ and PPARδ each bind to a PPAR response element close to the RGS5 promoter. RGS5 expression in nontransgenic MA was induced after activation of either PPARγ or PPARδ, an effect that was markedly blunted by DN PPARγ. Conclusions: We conclude that RGS5 in smooth muscle is a PPARγ and PPARδ target, which when activated blunts angiotensin II–mediated activation of protein kinase C, and preserves the large conductance Ca 2+ -activated K + channel activity, thus providing tight control of myogenic tone in the microcirculation.