The influence of erbium:yttrium-aluminum-garnet laser ablation with variable pulse width on morphology and microleakage of composite restorations

The objective of this study was to evaluate the influence of various pulse widths with different energy parameters of erbium:yttrium-aluminum-garnet (Er:YAG) laser (2.94 mu m) on the morphology and microleakage of cavities restored with composite resin. Identically sized class V cavities were prepared on the buccal surfaces of 54 bovine teeth by high-speed drill (n = 6, control, group 1) and prepared by Er:YAG laser (Fidelis 320A, Fotona, Slovenia) with irradiation parameters of 350 mJ/ 4 Hz or 400 mJ/2 Hz and pulse width: group 2, very short pulse (VSP); group 3, short pulse (SP); group 4, long pulse (LP); group 5, very long pulse (VLP). All cavities were filled with composite resin (Z-250-3 M), stored at 37A degrees C in distilled water, polished after 24 h, and thermally stressed (700 cycles/5-55A degrees C). The teeth were impermeabilized, immersed in 50% silver nitrate solution for 8 h, sectioned longitudinally, and exposed to Photoflood light for 10 min to reveal the stain. The leakage was evaluated under stereomicroscope by three different examiners, in a double-blind fashion, and scored (0-3). The results were analyzed by Kruskal-Wallis test (P > 0.05) and showed that there was no significant differences between the groups tested. Under scanning electron microscopy (SEM) the morphology of the cavities prepared by laser showed irregular enamel margins and dentin internal walls, and a more conservative pattern than that of conventional cavities. The different power settings and pulse widths of Er:YAG laser in cavity preparation had no influence on microleakage of composite resin restorations.

Pulp Vitality in Patients with Intraoral and Oropharyngeal Malignant Tumors Undergoing Radiation Therapy Assessed by Pulse Oximetry

Introduction: The aim of this study was to evaluate pulp oxygenation levels (%SpO(2)) in patients with malignant intraoral and oropharyngeal tumors treated by radiotherapy (RT). Methods: Pulp oxygenation levels were measured by pulse oximetry. Twenty patients were selected, and two teeth of each participant (n = 40) were analyzed, regardless of the quadrant and the area irradiated, at four different time points: TP1, before RI; TP2, at the beginning of RI with radiation doses between 30 and 35 Gy; TP3, at the end of RI with radiation dose! between 60 and 70 Gy; and TP4, 4 to 5 months after the beginning of cancer treatment. Results: Mean %SpO(2) at the different time points were 93% (TP1), 83% (TP2), 77% (TP3), and 85% (TP4). The Student`s t test showed statistically significant differences between TP1 and TP2 (P < .01), TP3 (P <.01), and TP4 (P <.01). TP3 was also statistically significantly different when compared with TP2 (P <.01) and TP4 (P <.01). No statistically significant difference could be observed between TP2 and TP4. Conclusion`s: Because the mean %SpO(2) before RI was greater than during and after therapy and values obtained 4 to 5 months after the beginning of RI were close to the initiation of RI, pulp tissue may be able to regain normal blood flow after RT. If the changes in the microcirculation of the dental pulp were indeed transitory, preventive endodontic treatment or extraction in patients who are currently undergoing or recently received RI and who show negative signs of pulp sensitivity may rot be necessary for pulpal reasons. (J Endod 2011;37:1197-1200)

Determination of pulp vitality in vivo with pulse oximetry

Aim To evaluate the use of pulse oximetry as a test for pulp vitality, by comparing in the same patient, the levels of oxygen saturation of the index finger and of the maxillary central incisor and canine teeth without clinically detectable pulp inflammation. Methodology Seventeen male and female patients aged between 26 and 38 years participated and a total of 32 maxillary central incisor and 32 canine teeth were analysed. Selection criteria required the teeth to have healthy crowns, or with restorations no more than 2 mm in diameter and no clinical and radiographical signs or symptoms of pulp or periapical inflammatory changes. The negative control group consisted of 10 root filled teeth. Measurements were first taken from the index finger of patients. Their teeth were then subjected to a thermal test with refrigerant gas and then to a vitality test with pulse oximetry. Data were analysed by Pearson`s and paired t-tests. Results There were no significant statistical correlations between blood oxygen levels in the index finger and in the teeth of the patient (P > 0.05). There was a statistically significant difference in the oxygen levels between the two tooth groups studied and the index finger (P <= 0.002). Mean oxygen values in the index finger of patients were 95% (SD = 1.6), oxygen values in the maxillary central incisor were 91.29% (SD = 2.61) and mean oxygen values in maxillary canine were 90.69% (SD = 2.71). Conclusion The method determined consistently the level of blood oxygen saturation of the pulp in maxillary central incisor and canine teeth and can therefore be used for pulp vitality testing. Further studies are required to assess the effectiveness and validity of pulse oximetry in determining pulp vitality in traumatized teeth.

A possible mechanism of low molecular weight protein tyrosine phosphatase (LMW-PTP) activity modulation by glutathione action during human osteoblast differentiation

Objective: Low molecular weight protein tyrosine phosphatases (LMW-PTPs) are a family of enzymes strongly involved in the regulation of cell growth and differentiation. Since there is no information concerning the relationship between osteoblastic differentiation and LMW-PTP expression/activity, we investigated its involvement during human osteoblast-like cells (hFOB 1.19) differentiation. It is known that LMW-PTP is regulated by an elegant redox mechanism, so we also observed how the osteoblastic differentiation affected the reduced glutathione levels. Design: hFOB 1.19 cells were cultured in DMEM/F12 up to 35 days. The osteoblast phenotype acquisition was monitored by measuring alkaline phosphatase activity and mineralized nodule formation by Von Kossa staining. LMW-PTP activity and expression were measured using the p-nitrophenylphosphate as substrate and Western blotting respectively. Crystal violet assay determined the cell number in each experimental point. Glutathione level was determined by both HPLC and DNTB assays. Results: LMW-PTP modulation was coincident with the osteoblastic differentiation biomarkers, such as alkaline phosphatase activity and presence of nodules of mineralization in Vitro. Likewise LMW-PTP, the reduced glutathione-dependent microenvironment was modulated during osteoblastic differentiation. During this process, LMW-PTP expression/activity, as well as alkaline phosphatase and glutathione increased progressively up to the 21st day (p < 0.001) of culturing, decreasing thereafter. Conclusions: Our results clearly suggest that LMW-PTP expression/activity was rigorously modulated during osteoblastic differentiation, possibly in response to the redox status of the cells, since it seems to depend on suitable levels of reduced glutathione. in this way, we pointed out LMW-PTP as an important signaling molecule in osteoblast biology and bone formation. (C) 2009 Elsevier Ltd. All rights reserved.

The broad effects of the functional IL-10 promoter-592 polymorphism: modulation of IL-10, TIMP-3, and OPG expression and their association with periodontal disease outcome

Periodontal diseases are infectious diseases, in which periodontopathogens trigger chronic inflammatory and immune responses that lead to tissue destruction. It occurs through the generation of metalloproteinases and the activation of bone resorption mechanisms. Anti-inflammatory cytokines such as IL-10 seem to attenuate periodontal tissue destruction through the induction of tissue inhibitors of metalloproteinases (TIMPs) and the inhibitor of osteoclastogenesis osteoprotegerin (OPG). A high individual variation in levels of IL-10 mRNA is verified in periodontitis patients, which is possibly determined by genetic polymorphisms. In this study, the IL-10 promoter -592C/A single nucleotide polymorphism ( SNP), which is associated with a decrease in IL-10 production, was analyzed by RFLP in 116 chronic periodontitis (CP) patients and 173 control (C) subjects, and the IL-10, TIMPs, and OPG mRNA expression levels in diseased gingival tissues were determined by real-time-PCR. The IL-10-592 SNP CA (P=0.0012/OR=2.4/CI:1.4-4.1), AA (P=0.0458/OR=2.3/CI:1.1-4.9), and CA+AA (P=0.0006/OR=2.4/CI: 1.4-3.4) genotypes and the allele A (P=0.0036/OR=1.7/CI:1.2-2.4) were found to be significantly more prevalent in the CP group when compared with control subjects. Both CA and AA genotypes were associated with lower levels of IL-10, TIMP-3, and OPG mRNA expression in diseased periodontal tissues and were also associated with disease severity as mean pocket depth. Taken together, the results presented here demonstrate that IL10-592 SNP is functional in CP, being associated with lower levels of IL-10 mRNA expression, which is supposed to consequently decrease the expression of the downstream genes TIMP-3 and OPG, and influence periodontal disease outcome. J. Leukoc. Biol. 84: 1565-1573; 2008.

Modulation of dopamine uptake by nitric oxide in cultured mesencephalic neurons

Strong evidence obtained from in vivo and ex-vivo studies suggests the existence of interaction between dopaminergic and nitrergic systems. Some of the observations suggest a possible implication of nitric oxide (NO) in dopamine (DA) uptake mechanism. The present work investigated the interaction between both systems by examining the effect of an NO donor, sodium nitroprusside (SNP), associated with the indirect DA agonist, amphetamine (AMPH) on tritiated DA uptake in cultures of embryonic mesencephalic neurons. Consistent with the literature, both AMPH (1, 3 and 10 mu M) and SNP (300 mu M and 1 mM) inhibited DA uptake in a dose-dependent manner. In addition, the inhibition of DA uptake by AMPH (1 and 3 mu M) was significantly increased by the previous addition of SNP (300 mu M). The implication of NO in this interaction was supported by the fact that the free radical scavenger N-acetyl-L-Cysteine (500 mu M) significantly increased DA uptake and completely abolished the effect of SNP, leaving unaffected that from AMPH on DA uptake. Further, double-labeling immunohistochemistry showed the presence of tyrosine hydroxylase-(TH, marker for dopaminergic neurons) and neuronal NO synthase- (nNOS, marker for NO containing neurons) expressing neurons in mesencephalic cultures. Some dopaminergic neurons also express nNOS giving further support for a pre-synaptic interaction between both systems. This is the first work demonstrating in mesencephalic cultured neurons a combined effect of an NO donor and an indirect DA agonist on specific DA uptake. (C) 2008 Elsevier B.V. All rights reserved.

Serotonergic mechanisms on breathing modulation in the rat locus coeruleus

The locus coeruleus (LC) is a noradrenergic nucleus that plays an important role in the ventilatory response to hypercapnia. This nucleus is densely innervated by serotonergic fibers and contains high density of serotonin (5-HT) receptors, including 5-HT(1A) and 5-HT(2). We assessed the possible modulation of respiratory response to hypercapnia by 5-HT, through 5-HT(1A) and 5-HT(2) receptors, in the LC. To this end, we determined the concentrations of 5-HT and its metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) in the LC after hypercapnic exposure. Pulmonary ventilation (V(E), plethysmograph) was measured before and after unilateral microinjection (100 nL) of WAY-100635 (5-HT(1A) antagonist, 5.6 and 56 mM), 8-OHDPAT (5-HT(1A/7) agonist, 7 and 15 mM), Ketanserin (5-HT(2A) antagonist, 3.7 and 37 mM), or (+/-)-2,5-dimethoxy-4-iodoamphetaminehydrochloride (DOI; 5-HT(2A) agonist, 6.7 and 67 mM) into the LC, followed by a 60-min period of 7% CO(2) exposure. Hypercapnia increased 5-HTIAA levels and 5-HIAA/5-HT ratio within the LC. WAY-100635 and 8-OHDPAT intra-LC decreased the hypercapnic ventilatory response due to a lower tidal volume. Ketanserin increased CO(2) drive to breathing and DOI caused the opposite response, both acting on tidal volume. The current results provide evidence of increased 5-HT release during hypercapnia in the LC and that 5-HT presents an inhibitory modulation of the stimulatory role of LC on hypercapnic ventilatory response, acting through postsynaptic 5-HT(2A) receptors in this nucleus. In addition, hypercapnic responses seem to be also regulated by presynaptic 5-HT(1A) receptors in the LC.

Nitric oxide modulation of methylphenidate-induced disruption of prepulse inhibition in Swiss mice

Drugs that facilitate dopaminergic neurotransmission induce cognitive and attentional deficits which include inability to filter sensory input measured by prepulse inhibition (PPI) Methylphenidate, an amphetamine analog is used in the treatment of attention deficit hyperactivity disorder Given that nitric oxide (NO) modulates dopamine effect our aim is to analyze the nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) inhibitors effect on PPI disruption induced by methylphenidate The inhibitors effects were compared to those produced by haloperidol and clozapine Male Swiss mice received a first I p. Injection (one hour before testing), of either saline, or N(G) nitro L-arginine (10, 40 or 90 mg/kg) or 7-Nitroindazole (3, 10, 30 or 60 mg/kg). or oxadiazolo-quinoxalin (5 or 10 mg/kg). or haloperidol (1 mg/kg), or clozapine (5 mg/kg) Thirty min later mice received the second injection of either saline or methylphenidate (20 or 30 mg/kg) or amphetamine (5 or 10 mg/kg). One group of mice received intracerebroventricular 7-Nitroindazole (50 or 100 nM) followed by systemic administration of saline or methylphenidate (30 mg/kg) The results revealed a methylphenidate dose-dependent disruption of PPI comparable to amphetamine. The effect was prevented by either nitric oxide synthase or guanilate cyclase inhibitors or clozapine or haloperidol In conclusion, methylphenidate induced a dose-dependent PPI disruption in Swiss mice modulated by dopamine and NO/sGC. The results corroborate the hypothesis of dopamine and NO interacting to modulate sensorimotor gating through central nervous system. It may be useful to understand methylphenidate and other psychostimulants effects (C) 2009 Elsevier B.V All rights reserved

Influence of pulse repetition rate of Er : YAG laser and dentin depth on tensile bond strength of dentin-resin interface

The study evaluated the in vitro influence of pulse-repetition rate of Er:YAG laser and dentin depth on tensile bond strength of dentin-resin interface. Dentin surfaces of buccal or lingual surfaces from human third molars were submitted to tensile test in different depths (superficial, 1.0 and 1.5 mm) of the same dental area, using the same sample. Surface treatments were acid conditioning solely (control) and Er:YAG laser irradiation (80 mJ) followed by acid conditioning, with different pulse-repetition rates (1, 2, 3, or 4 Hz). Single bond/Z-250 system was used. The samples were stored in distilled water at 37 degrees C for 24 h, and then the first test (superficial dentine) was performed. The bond failures were analyzed. Following, the specimens were identified, grounded until 1.0- and 1.5-mm depths, submitted again to the treatments and to the second and, after that, to third-bond tests on a similar procedure and failure analysis. ANOVA and Tukey test demonstrated a significant difference (p < 0.001) for treatment and treatment X depth interaction (p < 0.05). The tested depths did not show influence (p > 0.05) on the bond strength of dentin-resin interface. It may be concluded that Er:YAG laser with 1, 2, 3, or 4 Hz combined with acid conditioning did not increase the resin tensile bond strength to dentin, regardless of dentin depth. (C) 2007 Wiley Periodicals, Inc.

Influence of energy and pulse repetition rate of Er : YAG laser on enamel ablation ability and morphological analysis of the laser-irradiated surface

Objective: To assess the influence of energy and pulse repetition rate of Er:YAG laser on the enamel ablation ability and substrate morphology. Methods: Fifteen crowns of molars were sectioned in four fragments, providing 60 samples, which were ground to flatten the enamel surface. The initial mass was obtained by weighing the fragments. The specimens were hydrated for I h, fixed, and a 3-mm-diameter area was delimited. Twelve groups were randomly formed according to the combination of laser energies (200, 250, 300, or 350 mJ) and pulse repetition rates (2, 3, or 4 Hz). The final mass was obtained and mass loss was calculated by the difference between the initial and final mass. The specimens were prepared for SEM. Data were submitted to ANOVA and Scheffe test. Results: The 4 Hz frequency resulted in higher mass loss and was statistically different from 2 and 3 Hz (p < 0.05). The increase of frequency produced more melted areas, cracks, and unselective and deeper ablation. The 350 mJ energy promoted greater mass loss, similar to 300 mJ. Conclusions: The pulse repetition rate influenced more intensively the mass loss and morphological alteration. Among the tested parameters, 350 mJ/3 Hz improved the ability of enamel ablation with less surface morphological alterations. (C) 2007 Wiley Periodicals, Inc. J Biomed Mater Res.

Ovarian-Steroid Modulation of Locus Coeruleus Activity in Female Rats: Involvement in Luteinising Hormone Regulation

The noradrenergic nucleus locus coeruleus (LC) has been reported to regulate luteinising hormone (LH) secretion in female rats. Both oestrogen and progestin receptors have been demonstrated in LC neurones, suggesting that these cells are possibly responsive to variations in circulating levels of ovarian steroids. We therefore evaluated changes in the activity of LC neurones during the oestrous cycle and after ovarian-steroid treatment in ovariectomised (OVX) rats, as determined by immunoreactivity to Fos-related antigens (FRA), which comprises all of the known members of the Fos family. Effects of ovarian steroids on the firing rate of LC neurones were also determined in a slice preparation. The number of FRA/tyrosine hydroxylase (TH)-immunoreactive (ir) neurones in the LC increased from 14.00-16.00 h on pro-oestrus, coinciding with the onset of the LH surge and rise in plasma progesterone. FRA immunoreactivity was unaltered during dioestrus. Oestradiol-treated OVX rats (OVX+E) displayed marked reduction in FRA/TH-ir neurones in LC compared to oil-treated OVX rats. Accordingly, oestradiol superfusion significantly reduced the spontaneous firing rate of LC neurones in slices from OVX rats. Compared to OVX+E, oestradiol-treated rats injected with progesterone at 08.00 h (OVX+EP) exhibited higher number of FRA/TH-ir neurones in the LC at 10.00 h and 16.00 h, and great amplification of the LH surge. Bath application of progesterone significantly increased the spontaneous firing rate of OVX+E LC neurones. Our data suggest that ovarian steroids may physiologically modulate the activity of LC neurones in females, with possible implications for LH secretion. Moreover, oestradiol and progesterone appear to exert opposite and complementary effects (i.e. whereas oestradiol inhibits, progesterone, after oestradiol priming, stimulates LC activity).

Exercise improves cardiovascular control in a model of dislipidemia and menopause

The present study investigated the effects of exercise training on arterial pressure, baroreflex sensitivity, cardiovascular autonomic control and metabolic parameters on female LDL-receptor knockout ovariectomized mice. Mice were divided into two groups: sedentary and trained. Trained group was submitted to an exercise training protocol. Blood cholesterol was measured. Arterial pressure (AP) signals were directly recorded in conscious mice. Baroreflex sensitivity was evaluated by tachycardic and bradycardic responses to AP changes. Cardiovascular autonomic modulation was measured in frequency (FFT) and time domains. Maximal exercise capacity was increased in trained as compared to sedentary group. Blood cholesterol was diminished in trained mice (191 +/- 8 mg/dL) when compared to sedentary mice (250 +/- 9 mg/dL, p<0.05). Mean AP and HR were reduced in trained group (101 +/- 3 mmHg and 535 +/- 14 bpm, p<0.05) when compared with sedentary group (125 +/- 3 mmHg and 600 +/- 12 bpm). Exercise training induced improvement in bradycardic reflex response in trained animals (-4.24 +/- 0.62 bpm/mmHg) in relation to sedentary animals (-1.49 +/- 0.15 bpm/mmHg, p<0.01); tachycardic reflex responses were similar between studied groups. Exercise training increased the variance (34 +/- 8 vs. 6.6 +/- 1.5 ms(2) in sedentary, p<0.005) and the high-frequency band (HF) of the pulse interval (IP) (53 +/- 7% vs. 26 +/- 6% in sedentary, p<0.01). It is tempting to speculate that results of this experimental study might represent a rationale for this non-pharmacological intervention in the management of cardiovascular risk factors in dyslipidemic post-menopause women. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Clock Genes, Melanopsins, Melatonin, and Dopamine Key Enzymes and Their Modulation by Light and Glutamate in Chicken Embryonic Retinal Cells

The avian circadian system is composed of the retina, the mammalian homolog region of the suprachiasmatic nucleus (SNC), and the pineal gland. The retina, itself, displays many rhythmic physiological events, such as movements of photoreceptor cells, opsin expression, retinal reisomerization, and melatonin and dopamine production and secretion. Altogether, these rhythmic events are coordinated to predict environmental changes in light conditions during the day, optimizing retina function. The authors investigated the expression pattern of the melanopsin genes Opn4x and Opn4m, the clock genes Clock and Per2, and the genes for the key enzymes N-Acetyltransferase and Tyrosine Hidroxylase in chicken embryo dispersed retinal cells. Primary cultures of chicken retina from 8-day-old embryos were kept in constant dark (DD), in 12-h light/12-h dark (12L:12D), in 12L:12D followed by DD, or in DD in the absence or presence of 100 mu M glutamate for 12 h. Total RNA was extracted throughout a 24-h span, every 3 h starting at zeitgeber time 0 (ZT0) of the 6th day, and submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) followed by quantitative PCR (qPCR) for mRNA quantification. The data showed no rhythmic pattern of transcription for any gene in cells kept in DD. However under a light-dark cycle, Clock, Per2, Opn4m, N-Acetyltransferase, and Tyrosine Hydroxylase exhibited rhythmic patterns of transcription. In DD, 100 mu M glutamate was able to induce rhythmic expression of Clock, strongly inhibited the expression of Tyrosine Hydroxylase, and, only at some ZTs, of Opn4x and Opn4m. The neurotransmitter had no effect on Per2 and N-Acetyltransferase transcription. The authors confirmed the expression of the protein OPN4x by immunocytochemistry. These results suggest that chicken embryonic retinal cells contain a functional circadian clock, whose synchronization requires light-dark cycle or glutamate stimuli. (Author correspondence: amdlcast@ib.usp.br).

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

Phenotypic modulation of cultured vascular smooth muscle cells: a functional analysis focusing on MLC and ERK1/2 phosphorylation

Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.