[Effects of Different Nutritional Scoring Systems on Prognosis of Elderly Patients with Multiple Myeloma].

OBJECTIVE: To analyze the prognostic nutritional index (PNI), controlling nutritional status (CONUT) and fibrinogen/albumin ratio (FAR) levels in elderly patients with multiple myeloma (MM) and their prognostic impact. METHODS: The clinical data of 74 elderly MM patients diagnosed in Gansu Provincial Hospital from January 2020 to July 2022 were retrospectively analyzed. The optimal cut-off values for PNI, CONUT score and FAR were obtained by receiver operating characteristic (ROC) curve, which were used for grouping patients. The correlation of above three indexes with clinical parameters such as sex, serum calcium (Ca), ß2-microglobulin (ß2-MG), serum creatinine (Cr) in elderly MM patients were analyzed. The survival rates of patients with different levels of each index were compared. Univariate and multivariate analysis of the impact of clinical indicators on the prognosis of patients were performed. RESULTS: The optimal cut-off values for PNI, CONUT score and FAR were 39.775, 3.5 and 0.175, respectively, according to which the patients were divided into high and low group. Statistical analysis showed that there were significant differences in albumin level among different groups (all P < 0.05). In addition, there was a significant difference in hemoglobin between high-PNI group and low-PNI group (P < 0.05), while in sex distribution between high-FAR and low-FAR group (P < 0.05). The survival rate of elderly MM patients with increased PNI, decreased CONUT score and FAR was higher (all P < 0.05). Univariate and multivariate analysis showed that ß2-MG, Cr, PNI, CONUT score and FAR were independent prognostic factors for elderly MM patients. CONCLUSION: PNI, CONUT score and FAR are related to some clinical indicators of elderly MM patients, and have an impact on the prognosis.

[Efficacy and Safety of Bendamustine in the Conditioning Regiment for Autologous Hematopoietic Stem Cell Transplantation in Patients with Lymphoma].

OBJECTIVE: To investigate the clinical efficacy and safety of bendamustine in the conditioning regimen for autologous stem cell transplantation in patients with lymphoma. METHODS: The clinical data of 35 patients with lymphoma, including 5 patients of Hodgkin lymphoma and 30 patients of non-Hodgkin lymphoma, who underwent autologous stem cell transplantation after pretreatment with BeEAM regimen from January 2020 to June 2022 in Gansu Provincial Hospital were retrospectively analyzed. Hematopoietic reconstitution, disease outcome after transplantation and the side effects were analyzed. RESULTS: All 35 patients achieved hematopoietic reconstitution after AHSCT，the median time to neutrophil engraftment was 11 (8-15) days, and the median time to platelet engraftment was 12 (9-17) days. Among the 35 patients, 4 patients died at the end of follow-up, including 3 patients died of lymphoma recurrence or progression and 1 patient died of cerebral hemorrhage. Among 34 patients, 30 had no disease progression at the end of follow-up. The OS rates of patients at 12 and 24 months after transplantation were 90.97% and 90.97%, respectively. The 12 and 24 months PFS rates were 89.64% and 84.92%, respectively. Thiry-five patients underwent grade 3-4 bone marrow suppression. The non-hematological toxicity of BeEAM pretreatment regimen mainly included nausea, vomiting, diarrhea, and oral mucositis, 35 patients experienced nausea and vomiting, but only 4 patients had grade 3-4 nausea and vomiting. Eight patients experienced Grade 1-2 diarrhea. Oral mucositis occurred in 12 patients, including 1 patient of grade 3 oral mucositis. One patient with grade 3 oral mucositis also had grade 3-4 hypokalemia and hypon atremia. 8.6% of patients experienced Grade 1-2 abnormal liver and kidney function. An addition, infectious fever occurred in 18 patients during neutropenia. All patients improved after symptomatic treatment, and there were no transplant-related death. CONCLUSION: Bendamustine as a pretreatment regimen for autologous stem cell transplantation in lymphoma is effective, and the side effects are tolerable.

[Clinical Features and Prognosis of Patients with Castleman's Disease].

OBJECTIVE: To analyze the clinical features and prognosis of patients with Castleman's disease (CD) and improve the diagnosis and treatment of CD. METHODS: Clinical data of patients diagnosed with CD by pathological biopsy in Gansu Provincial Hospital from January 2009 to November 2020 were retrospectively analyzed. According to clinical classification, the patients were divided into two groups: UCD (unicentric CD) group (n=20) and MCD (multicentric CD) group (n=9). The clinical manifestations, laboratory examination, treatment regimens, pathological examination and follow-up data were statistically analyzed. RESULTS: There were no significant differences in average age and gender ratio between UCD group and MCD group. In UCD patients, 80.0% were hyaline vascular type, and 20.0% were plasma cell type. In MCD patients, 33.3% were hyaline vascular type, 55.6% were plasma cell type, and 11.1% were mixed type. There was significant difference in pathological classification between the two groups (P=0.039). The UCD patients usually presented asymptomatic single lymph node enlargement with mild clinical symptoms, while the MCD patients were characterized by multiple superficial and deep lymph node enlargement throughout the body. The incidences of asthenia, splenomegaly, serous effusion in MCD group were higher than those in UCD group (P<0.05). Meanwhile, the incidences of anemia, hypoproteinemia, increased ESR, elevated serum globulin and elevated ß2-microglobulin were significantly higher than those in UCD group too (P<0.05). There was no significant difference in the incidences of abnormal WBC, PLT and elevated LDH between the two groups (P>0.05). Among 20 patients with UCD, 13 cases reached complete remission (CR), 1 case achieved partial remission (PR). Among 9 patients with MCD, 3 cases received CR and 4 cases received PR. CONCLUSION: Patients with CD requires pathological examination for diagnosis. Patients with UCD show mild clinical symptoms, good surgical treatment effect and good prognosis. Patients with MCD have diversified clinical manifestations and relatively poor prognosis, and these patients require comprehensive treatment.

[Clinical Characteristics and Prognosis of Patients with Primary Bone Diffuse Large B-Cell Lymphoma].

AbstractObjective: To investigate the clinical characteristics and prognosis of patients with primary bone diffuse large B-cell lymphoma. METHODS: The clinical data of 15 patients with primary diffuse large B-cell lymphoma treated in our hospital from January 2013 to December 2020 were collected, the clinical data and prognosis of the patients were analyzed retrospectively. RESULTS: The median age of the 15 patients was 59 (19-89) years old; among the patients, 7 were males and 8 were females, ostealgia was the initial symptom. The pathological types of the 15 patients were diffuse large B-cell lymphoma (DLBCL), 5 cases of Has type GCB subtype (5/15), and 10 cases of Non-GCB subtype (10/15). After 15 patients were diagnosed, 11 patients (11/15) received chemotherapy, 3 patients (3/15) received surgery, and 1 patient was untreated (1/15), median chemotherapy courses was 5 (1-9). 8 patients have achieved complete remission (8/15), 3 patients achieved partial remission (3/15), and 1 patient achieved stable disease (1/15), 1 patient was lost to follow-up (1/15), 1 patient was untreated (1/15), and 1 patient was progression of disease (1/15). Age, pathological subtype, sex, stage, ß2-MG level, LDH level, and the using of rituximab were not correlated with the complete remission rate of the patients(P>0.05), while the IPI score was correlated with the recent complete remission rate (P<0.05). The median follow-up time was 19 (1-38) months, 10 patients survived, in which 6 cases were still in complete remission, and the median time to progression-free survival was 15 (1-38) months. CONCLUSION: The first symptom of primary bone diffuse large B-cell lymphoma is bone pain, the main pathological subtype is Non-GCB, the optimal treatment is combined chemotherapy, and the IPI score is related to the prognosis of the treatment.

Study on a Novel Biodegradable and Antibacterial Fe-Based Alloy Prepared by Microwave Sintering.

This research produced a porous Fe-8 wt.% Cu alloy by microwave sintering in order to achieve (i) an increased biodegradation rate, and (ii) an antibacterial function. The Fe-8Cu alloy had higher density, hardness and degradation rate (about 2 times higher) but smaller and fewer surface pores, compared to the pure Fe. The Fe-8Cu alloy had a strong antibacterial function (the antibacterial rates against E. coli were up to 99.9%) and good biocompatibility. This work provides a novel approach of alloy design and processing to develop novel antibacterial Fe-based alloys.

[Coagulation Indexes and Their Prognostic Significance in Patients with Multiple Myeloma].

OBJECTIVE: To analyze the relationship between coagulation indexes and prognosis of patients with multiple myeloma (MM). METHODS: A total of 99 newly diagnosed MM patients treated in Gansu Provincial Hospital from October 2017 to October 2019 were enrolled. Plasma thromboplastin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-dimer (D-D), platelet (PLT), and other laboratory indexes were detected. The relationship between coagulation indexes and clinical characteristics of MM patients was analyzed. The differences in survival rates among MM patients with different levels of coagulation indexes were compared, and the effect of each clinical index on the prognosis of MM patients was analyzed by univariate and multivariate. RESULTS: Each coagulation index was correlated to sex, disease classification and stage, and ß2-MG level of MM patients. Disease stage and ß2-MG level were positively correlated with coagulation indexes, which means that patients with late-stage disease or high ß2-MG, IgA and IgG levels were more likely to develop into coagulation disorders (P<0.05). The survival rate of patients with MM exhibiting elevated PT, FIB, and D-D levels was significantly lower (P<0.05). Univariate and multivariate analysis showed that each coagulation index was the influencing factor on the follow-up outcomes of MM patients, in which FIB was an independent prognostic factor. CONCLUSION: Coagulation function is correlated with multiple clinical indicators of patients with MM and plays an important role in their prognosis.

[The Influence of Serum Homocysteine Level to the Prognosis of Newly Diagnosed Multiple Myeloma Patients and Analysis of Related Factors].

OBJECTIVE: To analyze the influence of serum homocysteine (Hcy) levels to the prognosis of newly diagnosed multiple myeloma (MM) patients, and to explore related factors affecting the prognosis of the patients. METHODS: The clinical pathological data of 180 newly diagnosed MM patients treated in our hospital from March 2013 to February 2015 were collected, and the patients were divided into high and low Hcy groups based on the median Hcy. The survival curves of the patients in the two groups were drawn to compare the differences of the survival; univariate and multivariate survival analysis was used to observe the influence of serum cysteine to the prognosis of newly diagnosed MM patients; the clinicopathological data of the patients with high and low Hcy in the two groups was compared, Pearson test was used to further analyzes the relationship between Hcy and different factors, and explores the related factors of Hcy affecting the prognosis of the patients. RESULTS: The median survival times of patients in the high and low Hcy groups were 32 (5-59) and 41 (7-71) months, respectively. The 3-year survival rate of the patients in high Hcy group was significantly lower than those in low Hcy group, and the difference shows statistically significant (P<0.05). The results of univariate survival analysis showed that the OS of newly diagnosed MM patients whom with advanced age, high bone disease grade, high-level bone marrow plasma cell count, LDH, C-reactive protein, Cr, ß2-MG, Hcy, low-level Hb, and ALB was significantly shortened (all P<0.05). The results of multivariate survival analysis showed that old age, high levels of bone marrow plasma cells, Cr, ß2-MG, low levels of Hb, and ALB were the independent risk factors shorting the overall survival (OS) time of newly diagnosed MM patients (all P<0.05), while Hcy showed no independent relation for the OS of patients (P>0.05). The Hb level of the patients in high Hcy group was significantly lower than those in low-Hcy group, while the LDH level was significantly higher than those in low Hcy group (all P<0.05). Pearson test results showed that serum Hcy and Hb showed negative correlation (r=-0.813, P<0.05), but it shows positive correlation with LDH (r=0.726, P<0.05). CONCLUSION: Serum Hcy level has a correlation trend with the survival of newly diagnosed MM, which is affected by factors such as Hb.

[Correlation of Body Mass Index, ABO Blood Group with Multiple Myeloma].

OBJECTIVE: To explore the correlation of body mass index (BMI), ABO blood group with multiple myeloma (MM). METHODS: 70 MM patients (MM group) and 10 healthy people (control group) were selected in the same period, the BMI of patients was calculated according to the height and weight, and the differences of BMI in 2 groups was compared. The distribution of age, sex, albumin (Alb), serum creatinine (Cr), hemoglobin (Hb) and red blood cell (RBC) in the two groups were analyzed. Differences in red blood cell distribution width (RDW), neutrophil/lymphocyte ratio (NLR), disease stage and lactate dehydrogenase (LDH) level, survival rate of MM patients with different BMI values and blood group were compared between two groups, and the differences in follow-up outcomes of MM patients were analyzed by univariate and multivariate logistic regression analysis. RESULTS: BMI level of MM patients was higher than that of control group (t=2.706, P＜0.01), but the difference of blood group was not significant (P＞0.05); The NLR value in obese patients was higher than that in non-obese patients, the staging was later and the Alb level was lower in obese patients than those in non-obese patients. the differences were statistically significant between obese patients and non-obese patients (P＜0.05). Univariate and multivariate analysis showed that BMI, Alb and LDH level could influence the follow-up outcome of MM patients, the patients with elevated BMI and LDH level had worse prognosis, while patients with elevated Alb had better prognosis. which means that all the three factors are independent factors affecting the prognosis of MM patrents. CONCLUSION: Increased BMI in MM patients can affect the outcome of follow-up, which is an independent influencing factor.

[Significance of Neutrophil/Lymphocyte Ratio in the Prognosis of Patients with Multiple Myeloma].

OBJECTIVE: To study the value of neutrophil/lymphocyte ratio (NLR) in the evaluation of prognosis of patients with multiple myeloma (MM). METHODS: NLR was calculated on the basis of the blood routine examination results of 65 patients with primary MM (MM group) and 83 persons receiving physical examination as control group, and the difference in 2 group was compared; moreover according to the median as threshold, the patients were divided into low NLR group (NLR＜2.34) and high NLR group (NLR≥2.34); the differences of age, sex, serum calcium ß2 (Ca), microglobulin (ß2-MG), albumin (Alb), serum creatinine (Cr), hemoglobin (Hb), red blood cell distribution width (RDW) and lactate dehydrogenase (LDH) in 2 group were analyzed, and the survival rate was compared between the high and low-NLR group. RESULTS: the NLR of MM patients was statistically significantly higher than that of the control group (z=-2.415, P＜0.05). Compared with the low NLR group, the ß2-MG and Cr levels of patients in the high NLR group seemed higher, but the difference was not statistically significant. The single-factor analysis showed that NLR, ß2-MG and Alb levels were risk factors for the prognosis of MM patients, and the multi-factor analysis showed that NLR and Alb level were independent risk factors influencing the prognosis of MM patients. CONCLUSION: NLR elevation in patients with primary diagnosis of MM indicates a poor prognosis, which is an independent risk factor affecting the prognosis.

Activation of Ras in the Vascular Endothelium Induces Brain Vascular Malformations and Hemorrhagic Stroke.

Cerebrovascular malformations (CVMs) affect approximately 3% of the population, risking hemorrhagic stroke, seizures, and neurological deficits. Recently Ras mutations have been identified in a majority of brain arterio-venous malformations. We generated an endothelial-specific, inducible HRASV12 mouse model, which results in dilated, proliferative blood vessels in the brain, blood-brain barrier breakdown, intracerebral hemorrhage, and rapid lethality. Organoid morphogenesis models revealed abnormal cessation of proliferation, abnormalities in expression of tip and stalk genes, and a failure to properly form elongating tubes. These defects were influenced by both hyperactive PI-3' kinase signaling and altered TGF-ß signaling. Several phenotypic changes predicted by the in vitro morphogenesis analysis were validated in the mouse model. These data provide a model of brain vascular malformations induced by mutant Ras and reveal insights into intersecting molecular mechanisms in the pathogenesis of brain vascular malformations.

Loss of NF1 expression in human endothelial cells promotes autonomous proliferation and altered vascular morphogenesis.

Neurofibromatosis is a well known familial tumor syndrome, however these patients also suffer from a number of vascular anomalies. The loss of NFl from the endothelium is embryonically lethal in mouse developmental models, however little is known regarding the molecular regulation by NF1 in endothelium. We investigated the consequences of losing NF1 expression on the function of endothelial cells using shRNA. The loss of NF1 was sufficient to elevate levels of active Ras under non-stimulated conditions. These elevations in Ras activity were associated with activation of downstream signaling including activation of ERK, AKT and mTOR. Cells knocked down in NF1 expression exhibited no cellular senescence. Rather, they demonstrated augmented proliferation and autonomous entry into the cell cycle. These proliferative changes were accompanied by enhanced expression of cyclin D, phosphorylation of p27(KIP), and decreases in total p27(KIP) levels, even under growth factor free conditions. In addition, NF1-deficient cells failed to undergo normal branching morphogenesis in a co-culture assay, instead forming planar islands with few tubules and branches. We find the changes induced by the loss of NF1 could be mitigated by co-expression of the GAP-related domain of NF1 implicating Ras regulation in these effects. Using doxycycline-inducible shRNA, targeting NF1, we find that the morphogenic changes are reversible. Similarly, in fully differentiated and stable vascular-like structures, the silencing of NF1 results in the appearance of abnormal vascular structures. Finally, the proliferative changes and the abnormal vascular morphogenesis are normalized by low-dose rapamycin treatment. These data provide a detailed analysis of the molecular and functional consequences of NF1 loss in human endothelial cells. These insights may provide new approaches to therapeutically addressing vascular abnormalities in these patients while underscoring a critical role for normal Ras regulation in maintaining the health and function of the vasculature.

Role of p47(phox) in regulating Cdc42GAP, vimentin, and contraction in smooth muscle cells.

Cdc42GAP (GTPase activating protein) has been shown to regulate smooth muscle contraction as well as cell motility, adhesion, proliferation, and apoptosis. We have recently shown that Cdc42GAP activity is suppressed in smooth muscle cells during contractile activation, which is reversed by inhibitors of reactive oxygen species (ROS). Because p47(phox), a regulatory subunit of NAD(P)H oxidase, has been implicated in smooth muscle signaling, we determined whether this subunit modulates Cdc42GAP activity in response to contractile stimulation. Transfection of smooth muscle cells with plasmids encoding short hairpin RNA (shRNA) against p47(phox), but not plasmids for luciferase shRNA, inhibited the expression of p47(phox). ROS production and the suppression of Cdc42GAP activity in response to stimulation with 5-hydroxytryptamine (5-HT) were attenuated in cells producing p47(phox) shRNA compared with cells producing luciferase shRNA. In contrast, the addition of hydrogen peroxide to p47(phox)-deficient cells suppressed the activity of Cdc42GAP. Furthermore, exposure to hydrogen peroxide led to a decrease in Cdc42GAP activity in an in vitro assay. Cdc42 activation, p21-activated kinase 1 (PAK1) phosphorylation at Thr-423 (an indication of PAK activation), and vimentin phosphorylation at Ser-56 in response to 5-HT activation were also attenuated in smooth muscle cells producing shRNA against p47(phox). The knockdown of p47(phox) inhibited smooth muscle contraction during stimulation with 5-HT but not hydrogen peroxide. These results suggest that the p47(phox) subunit of NAD(P)H oxidase may mediate the agonist-induced GAP suppression by controlling ROS generation in smooth muscle cells during agonist stimulation. p47(phox)-regulated GAP affects smooth muscle contraction likely through the Cdc42/PAK1/vimentin pathway.

Cdc42GAP, reactive oxygen species, and the vimentin network.

Cdc42GAP (GTPase-activating protein) has been implicated in the regulation of cell motility, adhesion, proliferation, and apoptosis. In this study, Cdc42GAP was cloned from smooth muscle tissues. Cdc42GAP, but not inactive R282A Cdc42GAP (alanine substitution at arginine-282), enhanced the GTP hydrolysis of Cdc42 in an in vitro assay. Furthermore, we developed an assay to evaluate the activity of Cdc42GAP in vivo. Stimulation of smooth muscle cells with 5-hydroxytryptamine (5-HT) resulted in the decrease in Cdc42GAP activity. The agonist-induced GAP suppression was reversed by reactive oxygen species inhibitors. Treatment with hydrogen peroxide also inhibited GAP activity in smooth muscle cells. Because the vimentin cytoskeleton undergoes dynamic changes in response to contractile activation, we evaluated the role of Cdc42GAP in regulating vimentin filaments. Smooth muscle cells were infected with retroviruses encoding wild-type Cdc42GAP or its R282A mutant. Expression of wild-type Cdc42GAP, but not mutant R282A GAP, inhibited the increase in the activation of Cdc42 upon agonist stimulation. Phosphorylation of p21-activated kinase (PAK) at Thr-423 (an indication of PAK activation), vimentin phosphorylation (Ser-56), partial disassembly and spatial remodeling, and contraction were also attenuated in smooth muscle cells expressing Cdc42GAP. Our results suggest that the activity of Cdc42GAP is regulated upon contractile activation, which is mediated by intracellular ROS. Cdc42GAP regulates the vimentin network through the Cdc42-PAK pathway in smooth muscle cells during 5-HT stimulation.

Abl knockout differentially affects p130 Crk-associated substrate, vinculin, and paxillin in blood vessels of mice.

Actin polymerization has recently emerged as an important cellular process that regulates smooth muscle contraction. Abelson tyrosine kinase (Abl) has been implicated in the regulation of actin dynamics and force development in vascular smooth muscle. In the present study, the systolic blood pressure was lower in Abl(-/-) knockout mice compared with wild-type mice. The knockout of Abl diminished the tyrosine phosphorylation of p130 Crk-associated substrate (CAS, an adapter protein associated with smooth muscle contraction) in resistance arteries upon stimulation with phenylephrine or angiotensin II. The agonist-elicited enhancement of F-actin-to-G-actin ratios in arteries assessed by fluorescent microscopy was also reduced in Abl(-/-) mice. It has been known that vinculin is a structural protein that links actin filaments to extracellular matrix via transmembrane integrins, whereas paxillin is a signaling protein associated with focal contacts mediating actin cytoskeleton remodeling. The expression of vinculin and paxillin at protein and messenger levels was lower in arterial vessels from Abl knockout mice. However, the agonist-induced increase in myosin phosphorylation was not attenuated in arteries from Abl knockout mice. These results indicate that Abl differentially regulates Crk-associated substrate, vinculin, and paxillin in arterial vessels. The Abl-regulated cellular process and blood pressure are independent of myosin activation in vascular smooth muscle.

Abl silencing inhibits CAS-mediated process and constriction in resistance arteries.

The tyrosine phosphorylated protein Crk-associated substrate (CAS) has previously been shown to participate in the cellular processes regulating dynamic changes in the actin architecture and arterial constriction. In the present study, treatment of rat mesenteric arteries with phenylephrine (PE) led to the increase in CAS tyrosine phosphorylation and the association of CAS with the adapter protein CrkII. CAS phosphorylation was catalyzed by Abl in an in vitro study. To determine the role of Abl tyrosine kinase in arterial vessels, plasmids encoding Abl short hairpin RNA (shRNA) were transduced into mesenteric arteries by chemical loading plus liposomes. Abl silencing diminished increases in CAS phosphorylation on PE stimulation. Previous studies have shown that assembly of the multiprotein compound containing CrkII, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and the Arp2/3 (Actin Related Protein) complex triggers actin polymerization in smooth muscle as well as in nonmuscle cells. In this study, Abl silencing attenuated the assembly of the multiprotein compound in resistance arteries on contractile stimulation. Furthermore, the increase in F/G-actin ratios (an index of actin assembly) and constriction on contractile stimulation were reduced in Abl-deficient arterial segments compared with control arteries. However, myosin regulatory light chain phosphorylation (MRLCP) elicited by contractile activation was not inhibited in Abl-deficient arteries. These results suggest that Abl may play a pivotal role in mediating CAS phosphorylation, the assembly of the multiprotein complex, actin assembly, and constriction in resistance arteries. Abl does not participate in the regulation of myosin activation in arterial vessels during contractile stimulation.

Dissociation of Crk-associated substrate from the vimentin network is regulated by p21-activated kinase on ACh activation of airway smooth muscle.

The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, ACh stimulation of tracheal smooth muscle strips increased the ratio of soluble to insoluble vimentin (an index of vimentin disassembly) in association with force development. ACh activation also induced vimentin phosphorylation at Ser(56) as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips on contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The ACh-elicited decrease in CAS distribution in cytoskeletal vimentin was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser(56) and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during ACh activation of tracheal smooth muscle tissues.

Critical role of vimentin phosphorylation at Ser-56 by p21-activated kinase in vimentin cytoskeleton signaling.

Phosphorylation and spatial reorganization of the vimentin network have been implicated in mediating smooth muscle contraction, cell migration, and mitosis. In this study, stimulation of cultured smooth muscle cells with 5-hydroxytryptamine (5-HT) induced PAK1 phosphorylation at Thr-423 (an indication of p21-activated kinase (PAK) activation). Treatment with PAK led to disassembly of wild-type (but not mutant S56A) vimentin filaments as assessed by an in vitro filament assembly assay. Furthermore, stimulation with 5-HT resulted in the dissociation of Crk-associated substrate (CAS; an adapter protein associated with smooth muscle force development) from cytoskeletal vimentin. Expression of mutant S56A vimentin in cells inhibited the increase in phosphorylation at Ser-56 and in the ratios of soluble to insoluble vimentin (an index of vimentin disassembly) and the dissociation of CAS from cytoskeletal vimentin in response to 5-HT activation compared with cells expressing wild-type vimentin. Because CAS may be involved in PAK activation, PAK phosphorylation was evaluated in cells expressing the S56A mutant. Expression of mutant S56A vimentin depressed PAK phosphorylation at Thr-423 induced by 5-HT. Expression of the S56A mutant also inhibited the spatial reorientation of vimentin filaments in cells in response to 5-HT stimulation. Our results suggest that vimentin phosphorylation at Ser-56 may inversely regulate PAK activation possibly via the increase in the amount of soluble CAS upon agonist stimulation of smooth muscle cells. Additionally, vimentin phosphorylation at this position is critical for vimentin filament spatial rearrangement elicited by agonists.