Mario Grossi

Elevated glucose levels promote contractile and cytoskeletal gene expression in vascular smooth muscle via Rho/protein kinase C and actin polymerization

Both type 1 and type 2 diabetes are associated with increased risk of cardiovascular disease. This is in part attributed to the effects of hyperglycemia on vascular endothelial and smooth muscle cells, but the underlying mechanisms are not fully understood. In diabetic animal models, hyperglycemia results in hypercontractility of vascular smooth muscle possibly due to increased activation of Rho-kinase. The aim of the present study was to investigate the regulation of contractile smooth muscle markers by glucose and to determine the signaling pathways that are activated by hyperglycemia in smooth muscle cells. Microarray, quantitative PCR, and Western blot analyses revealed that both mRNA and protein expression of contractile smooth muscle markers were increased in isolated smooth muscle cells cultured under high compared with low glucose conditions. This effect was also observed in hyperglycemic Akita mice and in diabetic patients. Elevated glucose activated the protein kinase C and Rho/Rho-kinase signaling pathways and stimulated actin polymerization. Glucose-induced expression of contractile smooth muscle markers in cultured cells could be partially or completely repressed by inhibitors of advanced glycation end products, L-type calcium channels, protein kinase C, Rho-kinase, actin polymerization, and myocardin-related transcription factors. Furthermore, genetic ablation of the miR-143/145 cluster prevented the effects of glucose on smooth muscle marker expression. In conclusion, these data demonstrate a possible link between hyperglycemia and vascular disease states associated with smooth muscle contractility.

Local inhibition of ornithine decarboxylase reduces vascular stenosis in a murine model of carotid injury

OBJECTIVES Polyamines are organic polycations playing an essential role in cell proliferation and differentiation, as well as in cell contractility, migration and apoptosis. These processes are known to contribute to restenosis, a pathophysiological process often occurring in patients submitted to revascularization procedures. We aimed to test the effect of α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase, on vascular cell pathophysiology in vitro and in a rat model of carotid arteriotomy-induced (re)stenosis. METHODS The effect of DFMO on primary rat smooth muscle cells (SMCs) and mouse microvascular bEnd.3 endothelial cells (ECs) was evaluated through the analysis of DNA synthesis, polyamine concentration, cell viability, cell cycle phase distribution and by RT-PCR targeting cyclins and genes belonging to the polyamine pathway. The effect of DFMO was then evaluated in arteriotomy-injured rat carotids through the analysis of cell proliferation and apoptosis, RT-PCR and immunohistochemical analysis of differential gene expression. RESULTS DFMO showed a differential effect on SMCs and on ECs, with a marked, sustained anti-proliferative effect of DFMO at 3 and 8 days of treatment on SMCs and a less pronounced, late effect on bEnd.3 ECs at 8 days of DFMO treatment. DFMO applied perivascularly in pluronic gel at arteriotomy site reduced subsequent cell proliferation and preserved smooth muscle differentiation without affecting the endothelial coverage. Lumen area in DFMO-treated carotids was 49% greater than in control arteries 4 weeks after injury. CONCLUSIONS Our data support the key role of polyamines in restenosis and suggest a novel therapeutic approach for this pathophysiological process.

Differential expression of proteins related to smooth muscle cells and myofibroblasts in human thoracic aortic aneurysm.

OBJECTIVES Increasing knowledge is required for a better comprehension of the etiology of thoracic aortic aneurysm (TAA). The aim of this study was to highlight the modulations in vascular cell phenotypes, including myofibroblasts (MFs), in human TAA specimens compared to healthy aortas. METHODS histology, RT-PCR and immunohistochemical analysis of a panel of molecules, including ED-A Fibronectin (Fn), smoothelin, CD34 and alpha-smooth muscle actin (alpha-SMA), selected on the basis of their informative potential as markers of smooth muscle cells (SMCs) and MF phenotypic modulation, were performed on all samples. RESULTS The media of TAAs was characterized by the absence of smoothelin, the unaltered expression of alpha-SMA accompanied by an alteration of its distribution pattern, and by the activated expression of the ED-A isoform of Fn. We found a concentration of round-shaped cells exclusively in the adventitia and in the perivascular tissue of TAAs, also rich in vasa vasorum, largely expressing alpha-SMA, while a sub-population also expressed ED-A Fn and CD34. CD34 was expressed by several cells in the intima of TAAs, together with cells expressing cytoplasmatic ED-A Fn and alpha-SMA in comparison to healthy aortas. CONCLUSION TAA specimens show an altered expression and localization of SMC and MF differentiation markers in comparison to healthy aortas, with possible implications on remodeling.

Inhibition of polyamine uptake potentiates the anti-proliferative effect of polyamine synthesis inhibition and preserves the contractile phenotype of vascular smooth muscle cells.

Increased vascular smooth muscle cell (VSMC) proliferation is a factor in atherosclerosis and injury‐induced arterial (re) stenosis. Inhibition of polyamine synthesis by α‐difluoro‐methylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase, attenuates VSMC proliferation with high sensitivity and specificity. However, cells can escape polyamine synthesis blockade by importing polyamines from the environment. To address this issue, polyamine transport inhibitors (PTIs) have been developed. We investigated the effects of the novel trimer44NMe (PTI‐1) alone and in combination with DFMO on VSMC polyamine uptake, proliferation and phenotype regulation. PTI‐1 efficiently inhibited polyamine uptake in primary mouse aortic and human coronary VSMCs in the absence as well as in the presence of DFMO. Interestingly, culture with DFMO for 2 days substantially (>95%) reduced putrescine (Put) and spermidine (Spd) contents without any effect on proliferation. Culture with PTI‐1 alone had no effect on either polyamine levels or proliferation rate, but the combination of both treatments reduced Put and Spd levels below the detection limit and inhibited proliferation. Treatment with DFMO for a longer time period (4 days) reduced Put and Spd below their detection limits and reduced proliferation, showing that only a small pool of polyamines is needed to sustain VSMC proliferation. Inhibited proliferation by polyamine depletion was associated with maintained expression of contractile smooth marker genes. In cultured intact mouse aorta, PTI‐1 potentiated the DFMO‐induced inhibition of cell proliferation. The combination of endogenous polyamine synthesis inhibition with uptake blockade is thus a viable approach for targeting unwanted vascular cell proliferation in vivo, including vascular restenosis. J. Cell. Physiol. 231: 1334–1342, 2016.

The Polyamine Pathway as a Potential Target for Vascular Diseases: Focus on Restenosis

Polyamines are organic polycations expressed by all living organisms, which are known to play an essential role in cell proliferation and differentiation. Recent studies revealed their involvement also in cell contractility and migration and in programmed cell death. These processes are known to contribute to restenosis, a pathophysiological process occurring in 10-20% of patients submitted to revascularization procedures. The advent of bare metal stents and of drug-eluting stents has significantly reduced but not eliminated the incidence of restenosis, which thus remains a clinically relevant problem. Despite the potential role of the polyamine pathway as a therapeutic target due to its involvement in proliferation, apoptosis and migration of vascular cells, experimental inhibition of polyamine synthesis and/or uptake has been poorly investigated in animal models of vascular disease. Here we review the current knowledge about molecular mechanisms related to polyamine functions, with particular reference to the role played by polyamines in vascular cell pathophysiology, together with experimental evidence obtained so far in animal models of (re)stenosis. We also evaluate the advantages of different routes of administration of polyamine synthesis/transport inhibitors and polyamine analogue molecules. Increasing knowledge about the molecular mechanisms and functions of polyamines is expected to shed new light on their potential role as a therapeutic target for restenosis reduction.

Assessing the contribution of thrombospondin-4 induction and ATF6α activation to endoplasmic reticulum expansion and phenotypic modulation in bladder outlet obstruction

Phenotypic modulation of smooth muscle cells is a hallmark of disease. The associated expansion of endoplasmic reticulum (ER) volume remains unexplained. Thrombospondin-4 was recently found to promote ATF6α activation leading to ER expansion. Using bladder outlet obstruction as a paradigm for phenotypic modulation, we tested if thrombospondin-4 is induced in association with ATF6α activation and ER expansion. Thrombospondin-4 was induced and ATF6α was activated after outlet obstruction in rodents. Increased abundance of spliced of Xbp1, another ER-stress sensor, and induction of Atf4 and Creb3l2 was also seen. Downstream of ATF6α, Calr, Manf, Sdf2l1 and Pdi increased as did ER size, whereas contractile markers were reduced. Overexpression of ATF6α, but not of thrombospondin-4, increased Calr, Manf, Sdf2l1 and Pdi and caused ER expansion, but the contractile markers were inert. Knockout of thrombospondin-4 neither affected bladder growth nor expression of ATF6α target genes, and repression of contractile markers was the same, even if ATF6α activation was curtailed. Increases of Xbp1s, Atf4 and Creb3l2 were similar. Our findings demonstrate reciprocal regulation of the unfolded protein response, including ATF6α activation and ER expansion, and reduced contractile differentiation in bladder outlet obstruction occurring independently of thrombospondin-4, which however is a sensitive indicator of obstruction.

PYK2 selectively mediates signals for growth versus differentiation in response to stretch of spontaneously active vascular smooth muscle.

Stretch of vascular smooth muscle stimulates growth and proliferation as well as contraction and expression of contractile/cytoskeletal proteins, all of which are also regulated by calcium‐dependent signals. We studied the role of the calcium‐ and integrin‐activated proline‐rich tyrosine kinase 2 (PYK2) in stretch‐induced responses of the rat portal vein loaded by a hanging weight ex vivo. PYK2 phosphorylation at Tyr‐402 was increased both by a 10‐min stretch and by organ culture with load over several days. Protein and DNA synthesis were reduced by the novel PYK2 inhibitor PF‐4594755 (0.5–1 μmol/L), while still sensitive to stretch. In 3‐day organ culture, PF‐4594755 caused maintained myogenic spontaneous activity but did not affect contraction in response to high‐K+ (60 mmol/L) or to α1‐adrenergic stimulation by cirazoline. Basal and stretch‐induced PYK2 phosphorylation in culture were inhibited by PF‐4594755, closely mimicking inhibition of non‐voltage‐dependent calcium influx by 2‐APB (30 μmol/L). In contrast, the L‐type calcium channel blocker, nifedipine (1 μmol/L) eliminated stretch‐induced but not basal PYK2 phosphorylation. Stretch‐induced Akt and ERK1/2 phosphorylation was eliminated by PF‐4594755. PYK2 inhibition had no effect on mRNA expression of several smooth muscle markers, and stretch‐sensitive SM22α synthesis was preserved. Culture of portal vein with the Ang II inhibitor losartan (1 μmol/L) eliminated stretch sensitivity of PYK2 and Akt phosphorylation, but did not affect mRNA expression of smooth muscle markers. The results suggest that PYK2 signaling functionally distinguishes effects of voltage‐ and non‐voltage‐dependent calcium influx. A small‐molecule inhibitor of PYK2 reduces growth and DNA synthesis but does not affect contractile differentiation of vascular smooth muscle.

Vascular smooth muscle cell proliferation depends on caveolin-1-regulated polyamine uptake

Much evidence highlights the importance of polyamines for VSMC (vascular smooth muscle cell) proliferation and migration. Cav-1 (caveolin-1) was recently reported to regulate polyamine uptake in intestinal epithelial cells. The aim of the present study was to assess the importance of Cav-1 for VSMC polyamine uptake and its impact on cell proliferation and migration. Cav-1 KO (knockout) mouse aortic cells showed increased polyamine uptake and elevated proliferation and migration compared with WT (wild-type) cells. Both Cav-1 KO and WT cells expressed the smooth muscle differentiation markers SM22 and calponin. Cell-cycle phase distribution analysis revealed a higher proportion of Cav-1 KO than WT cells in the S phase. Cav-1 KO cells were hyper-proliferative in the presence but not in the absence of extracellular polyamines, and, moreover, supplementation with exogenous polyamines promoted proliferation in Cav-1 KO but not in WT cells. Expression of the solute carrier transporters Slc7a1 and Slc43a1 was higher in Cav-1 KO than in WT cells. ODC (ornithine decarboxylase) protein and mRNA expression as well as ODC activity were similar in Cav-1 KO and WT cells showing unaltered synthesis of polyamines in Cav-1 KO cells. Cav-1 was reduced in migrating cells in vitro and in carotid lesions in vivo. Our data show that Cav-1 negatively regulates VSMC polyamine uptake and that the proliferative advantage of Cav-1 KO cells is critically dependent on polyamine uptake. We provide proof-of-principle for targeting Cav-1-regulated polyamine uptake as a strategy to fight unwanted VSMC proliferation as observed in restenosis.

Inhibition of polyamine formation antagonizes vascular smooth muscle cell proliferation and preserves the contractile phenotype.

The polyamines putrescine, spermidine and spermine play essential roles in cell proliferation and migration, two processes involved in the development of vascular disease. Thus, intervention with polyamine formation may represent a way to inhibit unwanted vascular smooth muscle cell (VSMC) proliferation. The aim of the present study was to assess the importance of polyamines for VSMC proliferation and vascular contractility. The rate‐limiting step in polyamine biosynthesis is catalysed by ornithine decarboxylase (ODC). Treatment with α‐difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, reduced DNA synthesis in primary rat VSMCs in a concentration‐dependent manner with an IC50 value of 100 μM. Moreover, DFMO reduced VSMC migration assessed in a scratch assay. The DFMO‐induced attenuation of VSMC proliferation was associated with lowered cellular amount of polyamines. The antiproliferative effect of DFMO was specific because supplementation with polyamines reversed the effect of DFMO on proliferation and normalized cellular polyamine levels. Isometric force recordings in cultured rat tail artery rings showed that DFMO counteracts the decrease in contractility caused by culture with foetal bovine serum as growth stimulant. We conclude that inhibition of polyamine synthesis by DFMO may limit the first wave of cell proliferation and migration, which occurs in the acute phase after vascular injury. Besides its antiproliferative effect, DFMO may prevent loss of the smooth muscle contractile phenotype in vascular injury.

Pyk2 inhibition promotes contractile differentiation in arterial smooth muscle

Modulation from contractile to synthetic phenotype of vascular smooth muscle cells is a central process in disorders involving compromised integrity of the vascular wall. Phenotype modulation has been shown to include transition from voltage‐dependent toward voltage‐independent regulation of the intracellular calcium level, and inhibition of non‐voltage dependent calcium influx contributes to maintenance of the contractile phenotype. One possible mediator of calcium‐dependent signaling is the FAK‐family non‐receptor protein kinase Pyk2, which is activated by a number of stimuli in a calcium‐dependent manner. We used the Pyk2 inhibitor PF‐4594755 and Pyk2 siRNA to investigate the role of Pyk2 in phenotype modulation in rat carotid artery smooth muscle cells and in cultured intact arteries. Pyk2 inhibition promoted the expression of smooth muscle markers at the mRNA and protein levels under stimulation by FBS or PDGF‐BB and counteracted phenotype shift in cultured intact carotid arteries and balloon injury ex vivo. During long‐term (24–96 hr) treatment with PF‐4594755, smooth muscle markers increased before cell proliferation was inhibited, correlating with decreased KLF4 expression and differing from effects of MEK inhibition. The Pyk2 inhibitor reduced Orai1 and preserved SERCA2a expression in carotid artery segments in organ culture, and eliminated the inhibitory effect of PDGF stimulation on L‐type calcium channel and large‐conductance calcium‐activated potassium channel expression in carotid cells. Basal intracellular calcium level, calcium wave activity, and store‐operated calcium influx were reduced after Pyk2 inhibition of growth‐stimulated cells. Pyk2 inhibition may provide an interesting approach for preserving vascular smooth muscle differentiation under pathophysiological conditions.