Involvement of Rho-Associated Coiled-Coil Containing Kinase (ROCK) in BCR-ABL1 Tyrosine Kinase Inhibitor Cardiovascular Toxicity.

Background: Second- and third-generation BCR-ABL1 tyrosine kinase inhibitors (TKIs) are associated with cardiovascular adverse events (CVAEs) in patients with Philadelphia chromosome-positive (Ph+) leukemia. Objectives: We hypothesized that second- and third-generation BCR-ABL1 TKIs may cause CVAEs through the activation of Rho-associated coiled-coil containing kinase (ROCK). Methods: Peripheral blood mononuclear cells from 53 Ph+ patients on TKIs and 15 control patients without Ph+ leukemia were assessed for ROCK activity through capillary electrophoresis (median follow-up = 26 months [Q1-Q3: 5-37 months]). We also investigated the effects of TKIs and ROCK on endothelial dysfunction in vitro, which could contribute to CVAEs. Results: Patients receiving second- and third-generation TKIs had 1.6-fold greater ROCK activity compared with patients receiving imatinib and control patients. Elevated ROCK activity was associated with an increased incidence of CVAEs in Ph+ leukemia patients. In endothelial cells in vitro, we found that dasatinib and ponatinib treatment led to changes in actin intensity and endothelial permeability, which can be reversed by pharmacologic inhibition of ROCK. Ponatinib led to decreased cell proliferation, but this was not accompanied by senescence. Dasatinib and ponatinib treatment led to phosphor-inhibition of endothelial nitric oxide synthase and decreased nitric oxide production. ROCK inhibition reversed endothelial permeability and endothelial nitric oxide synthase-related endothelial dysfunction. Imatinib and nilotinib induce phosphorylation of p190RhoGAP. Conclusions: Our findings suggest ROCK activity may be a prognostic indicator of CVAEs in patients receiving BCR-ABL1 TKIs. With further study, ROCK inhibition may be a promising approach to reduce the incidence of CVAEs associated with second- and third-generation BCR-ABL1 TKIs.

Vascular Stiffening Mediated by Rho-Associated Coiled-Coil Containing Kinase Isoforms.

Background The pathogenesis of vascular stiffening and hypertension is marked by non-compliance of vessel wall because of deposition of collagen fibers, loss of elastin fibers, and increased vascular thickening. Rho/Rho-associated coiled-coil containing kinases 1 and 2 (ROCK1 and ROCK2) have been shown to regulate cellular contraction and vascular remodeling. However, the role of ROCK isoforms in mediating pathogenesis of vascular stiffening and hypertension is not known. Methods and Results Hemizygous Rock mice (Rock1+/- and Rock2+/-) were used to determine the role of ROCK1 and ROCK2 in age-related vascular dysfunction. Both ROCK activity and aortic stiffness increased to a greater extent with age in wild-type mice compared with that of Rock1+/- and Rock2+/- mice. As a model for age-related vascular stiffening, we administered angiotensin II (500 ng/kg per minute) combined with nitric oxide synthase inhibitor, L-Nω-nitroarginine methyl ester (0.5 g/L) for 4 weeks to 12-week-old male Rock1+/- and Rock2+/- mice. Similar to advancing age, angiotensin II/L-Nω-nitroarginine methyl ester caused increased blood pressure, aortic stiffening, and vascular remodeling, which were attenuated in Rock2+/-, and to a lesser extent, Rock1+/- mice. The reduction of aortic stiffening in Rock2+/- mice was accompanied by decreased collagen deposition, relatively preserved elastin content, and less aortic wall hypertrophy. Indeed, the upregulation of collagen I by transforming growth factor-ß1 or angiotensin II was greatly attenuated in Rock2-/- mouse embryonic fibroblasts. Conclusions These findings indicate that ROCK1 and ROCK2 mediate both age-related and pharmacologically induced aortic stiffening, and suggest that inhibition of ROCK2, and to a lesser extent ROCK1, may have therapeutic benefits in preventing age-related vascular stiffening.

PPARγ activation improves the microenvironment of perivascular adipose tissue and attenuates aortic stiffening in obesity.

BACKGROUND: Obesity-related cardiovascular risk, end points, and mortality are strongly related to arterial stiffening. Current therapeutic approaches for arterial stiffening are not focused on direct targeting within the vessel. Perivascular adipose tissue (PVAT) surrounding the artery has been shown to modulate vascular function and inflammation. Peroxisome proliferator-activated receptor γ (PPARγ) activation significantly decreases arterial stiffness and inflammation in diabetic patients with coronary artery disease. Thus, we hypothesized that PPARγ activation alters the PVAT microenvironment, thereby creating a favorable environment for the attenuation of arterial stiffening in obesity. METHODS: Obese ob/ob mice were used to investigate the effect of PPARγ activation on the attenuation of arterial stiffening. Various cell types, including macrophages, fibroblasts, adipocytes, and vascular smooth muscle cells, were used to test the inhibitory effect of pioglitazone, a PPARγ agonist, on the expression of elastolytic enzymes. RESULTS: PPARγ activation by pioglitazone effectively attenuated arterial stiffening in ob/ob mice. This beneficial effect was not associated with the repartitioning of fat from or changes in the browning of the PVAT depot but was strongly related to improvement of the PVAT microenvironment, as evidenced by reduction in the expression of pro-inflammatory and pro-oxidative factors. Pioglitazone treatment attenuated obesity-induced elastin fiber fragmentation and elastolytic activity and ameliorated the obesity-induced upregulation of cathepsin S and metalloproteinase 12, predominantly in the PVAT. In vitro, pioglitazone downregulated Ctss and Mmp12 in macrophages, fibroblasts, and adipocytes-cell types residing within the adventitia and PVAT. Ultimately, several PPARγ binding sites were found in Ctss and Mmp12 in Raw 264.7 and 3T3-L1 cells, suggesting a direct regulatory mechanism by which PPARγ activation repressed the expression of Ctss and Mmp-12 in macrophages and fibroblasts. CONCLUSIONS: PPARγ activation attenuated obesity-induced arterial stiffening and reduced the inflammatory and oxidative status of PVAT. The improvement of the PVAT microenvironment further contributed to the amelioration of elastin fiber fragmentation, elastolytic activity, and upregulated expression of Ctss and Mmp12. Our data highlight the PVAT microenvironment as an important target against arterial stiffening in obesity and provide a novel strategy for the potential clinical use of PPARγ agonists as a therapeutic against arterial stiffness through modulation of PVAT function.

Peroxisome Proliferator-Activated Receptor γ Level Contributes to Structural Integrity and Component Production of Elastic Fibers in the Aorta.

Loss of integrity and massive disruption of elastic fibers are key features of abdominal aortic aneurysm (AAA). Peroxisome proliferator-activated receptor γ (PPARγ) has been shown to attenuate AAA through inhibition of inflammation and proteolytic degradation. However, its involvement in elastogenesis during AAA remains unclear. PPARγ was highly expressed in human AAA within all vascular cells, including inflammatory cells and fibroblasts. In the aortas of transgenic mice expressing PPARγ at 25% normal levels (Pparg(C) (/-) mice), we observed the fragmentation of elastic fibers and reduced expression of vital elastic fiber components of elastin and fibulin-5. These were not observed in mice with 50% normal PPARγ expression (Pparg(+/-) mice). Infusion of a moderate dose of angiotensin II (500 ng/kg per minute) did not induce AAA but Pparg(+/-) aorta developed flattened elastic lamellae, whereas Pparg(C/-) aorta showed severe destruction of elastic fibers. After infusion of angiotensin II at 1000 ng/kg per minute, 73% of Pparg(C/-) mice developed atypical suprarenal aortic aneurysms: superior mesenteric arteries were dilated with extensive collagen deposition in adventitia and infiltrations of inflammatory cells. Although matrix metalloproteinase inhibition by doxycycline somewhat attenuated the dilation of aneurysm, it did not reduce the incidence nor elastic lamella deterioration in angiotensin II-infused Pparg(C/-) mice. Furthermore, PPARγ antagonism downregulated elastin and fibulin-5 in fibroblasts, but not in vascular smooth muscle cells. Chromatin immunoprecipitation assay demonstrated PPARγ binding in the genomic sequence of fibulin-5 in fibroblasts. Our results underscore the importance of PPARγ in AAA development though orchestrating proper elastogenesis and preserving elastic fiber integrity.

Loss of Egr-1 sensitizes pancreatic ß-cells to palmitate-induced ER stress and apoptosis.

UNLABELLED: Pancreatic ß-cells are particularly susceptible to fatty-acid-induced endoplasmic reticulum (ER) stress and apoptosis. To understand how ß-cells sense fatty acid stimuli and translate into a long-term adaptive response, we investigated whether palmitic acid (PA) regulates early growth response-1 (Egr-1), an immediate-early transcription factor, which is induced by many environmental stimuli and implicated in cell proliferation, differentiation, and apoptosis. We found that Egr-1 was rapidly and transiently induced by PA in MIN6 insulinoma cells, which was accompanied by calcium influx and ERK1/2 phosphorylation. Calcium chelation and MEK1/2 inhibition blocked PA-induced Egr-1 upregulation, suggesting that PA induces Egr-1 expression through a calcium influx-MEK1/2-ERK1/2 cascade. Knockdown of Egr-1 increased PA-induced caspase-3 activation and ER stress markers and decreased PA-induced Akt phosphorylation and insulin secretion and signaling. Akt replenishment and insulin supplementation rescued PA-induced apoptosis in Egr-1 knockdown cells. These results suggest that the absence of Egr-1 loses its ability to couple the short-term insulin/Akt pathway to long-term survival adaptation. Finally, Egr-1-deficient mouse islets are more susceptible to ex vivo stimuli of apoptosis. In human pancreatic tissues, EGR1 expression correlated with expression of ER stress markers and anti-apoptotic gene. In conclusion, Egr-1 is induced by PA and further attempts to rescue ß-cells from ER stress and apoptosis through improving insulin/Akt signaling. Our study underscores Egr-1 as a critical early sensor in pancreatic ß-cells to translate fatty acid stimuli into a cellular adaptation mechanism. KEY MESSAGE: PA stimulates Egr-1 expression via a calcium influx-MEK1/2-ERK1/2-Elk-1 cascade. Egr-1 attenuates PA-induced ER stress and apoptosis. Egr-1 maintains Akt survival pathway to protect ß-cells from PA-induced apoptosis. Egr-1-deficient islets are prone to ex vivo stimuli of apoptosis. Human EGR1 expression correlates with genes for ER stress and anti-apoptosis.

Increased aortic stiffness and attenuated lysyl oxidase activity in obesity.

OBJECTIVE: One potential mechanism through which obesity exerts adverse effects on the vascular system is by increasing aortic stiffness, a change known to be predictive of increased cardiovascular mortality. The aim of this study was to investigate the pathophysiology that links obesity to aortic stiffening. APPROACH AND RESULTS: Obese (ob/ob) mice were used to examine physical, morphological, and molecular changes in the aorta in response to obesity. ob/ob mice had increased aortic pulse wave velocity and tissue rigidity. ob/ob aorta exhibited decreases of lysyl oxidase (LOX) activity and cross-linked elastin, and increases of elastin fragmentation and elastolytic activity. The aortas of ob/ob mice were surrounded by a significant amount of proinflammatory and pro-oxidative perivascular adipose tissue. In vitro studies revealed that the conditioned medium from differentiated adipocytes or the perivascular adipose tissue of ob/ob mice attenuated LOX activity. Furthermore, inhibition of LOX in wild-type lean mice caused elastin fragmentation and induced a significant increase in pulse wave velocity. Finally, we found that obese humans had stiffer arteries and lower serum LOX levels than do normal-weight humans. CONCLUSIONS: Our results demonstrated that obesity resulted in aortic stiffening in both humans and mice, and established a causal relationship between LOX downregulation and aortic stiffening in obesity.