S-Nitrosylation in endothelial cells contributes to tumor cell adhesion and extravasation during breast cancer metastasis.

BACKGROUND: Nitric oxide is produced by different nitric oxide synthases isoforms. NO activates two signaling pathways, one dependent on soluble guanylate cyclase and protein kinase G, and other where NO post-translationally modifies proteins through S-nitrosylation, which is the modification induced by NO in free-thiol cysteines in proteins to form S-nitrosothiols. High levels of NO have been detected in blood of breast cancer patients and increased NOS activity has been detected in invasive breast tumors compared to benign or normal breast tissue, suggesting a positive correlation between NO biosynthesis, degree of malignancy and metastasis. During metastasis, the endothelium plays a key role allowing the adhesion of tumor cells, which is the first step in the extravasation process leading to metastasis. This step shares similarities with leukocyte adhesion to the endothelium, and it is plausible that it may also share some regulatory elements. The vascular cell adhesion molecule-1 (VCAM-1) expressed on the endothelial cell surface promotes interactions between the endothelium and tumor cells, as well as leukocytes. Data show that breast tumor cells adhere to areas in the vasculature where NO production is increased, however, the mechanisms involved are unknown. RESULTS: We report that the stimulation of endothelial cells with interleukin-8, and conditioned medium from breast tumor cells activates the S-nitrosylation pathway in the endothelium to induce leukocyte adhesion and tumor cell extravasation by a mechanism that involves an increased VCAM-1 cell surface expression in endothelial cells. We identified VCAM-1 as an S-nitrosylation target during this process. The inhibition of NO signaling and S-nitrosylation blocked the transmigration of tumor cells through endothelial monolayers. Using an in vivo model, the number of lung metastases was inhibited in the presence of the S-nitrosylation inhibitor N-acetylcysteine (NAC), which was correlated with lower levels of S-nitrosylated VCAM-1 in the metastases. CONCLUSIONS: S-Nitrosylation in the endothelium activates pathways that enhance VCAM-1 surface localization to promote binding of leukocytes and extravasation of tumor cells leading to metastasis. NAC is positioned as an important tool that might be tested as a co-therapy against breast cancer metastasis.

Hipersensibilidad a corticoides y manejo en asma severa. A propósito de un caso / Hipersensitivity to corticosteroids and management of severe bronchial asthma. Case study

Resumen Las reacciones de hipersensibilidad a corticoides son raras en la población general, se dividen en dos categorías: Inmediatas, típicamente mediadas por Inmunoglobulina E (IgE), donde se incluye la anafilaxia luego de la administración de un fármaco en un corto período. Su prevalencia descrita es de 0,3-0,5%. Otra reacción es la 'no inmediata', que se manifiesta en un tiempo mayor de una hora después de la administración del fármaco. Se revisó la literatura con el objetivo de mejorar y aclarar el tratamiento en pacientes asmáticos que poseen esta condición. Se encontró que las vías posibles para generar estas reacciones son intranasal, aerosol por inhalador, oral y parenteral. Frente a esta condición se requiere una evaluación estrecha y detallada de la historia clínica, síntomas y reacciones secundarias al fármaco sospechoso. Finalmente, al momento de elegir tipo de corticoide a usar es primordial la seguridad del paciente logrando, además, el control de la enfermedad.

Hypersensitivity reactions to corticosteroids are rare in the general population, they fall into two categories: 'immediate', typically mediated by immunoglobulin E (IgE), which includes anaphylaxis after administration of a drug in a short period of time. Its reported prevalence is 0.3-0.5%. Another reaction is 'not immediate', which manifests itself in a time longer than one hour after the administration of the drug. We reviewed the literature with the aim of improving and clarifying the treatment in asthmatic patients with this condition. It was found that the possible routes to generate these reactions are intranasal, aerosol by inhaler, oral and parenteral. Facing this condition requires a close and detailed evaluation of the clinical history, symptoms and side reactions to the suspected drug. Finally, when choosing which corticosteroid to use, the patient's safety is paramount, and control of the disease is also essential.

Regulation of matrix contraction in chronic venous disease.

OBJECTIVE: The role of TGF-beta(1) in venous ulcer healing and the signalling cascades regulating dermal fibroblast function are poorly understood. To elucidate these processes, we hypothesized that TGF-beta(1) facilitates wound healing by increasing chronic venous insufficiency (CVI) induced matrix contraction via intracellular cross-talk between TGF-beta(1) and the ERK-1/2 MAP kinase signalling cascades. METHODS: Fibroblasts isolated from calf biopsies (LC) of patients with different severity of CVI (CEAP, Clinical Etiological Anatomical Pathological classes) were seeded into 200 microl collagen gels under isometric conditions. Fibroblasts from neonatal foreskins (HS68), non-CVI patients (NC), and the ipsilateral normal thigh of each CVI patient (LT) served as controls. Thirteen patients with CVI (class 2, n=5; class 4, n=5; class 6, n=3) and 2 non-CVI controls (NC, n=2) were included in the study. All experimental conditions were determined by dose-response and time-course experiments. Gels were cultured with/without 0.1 ng/ml TGF-beta(1) and with/without 50 microM PD98059 (MEK and downstream-MAPK inhibitor). Additional patient fibroblasts were transfected with constitutively active Ras (pCMV-Ras) or an empty vector (pCMV-beta) with/without 0.1 ng/ml TGF-beta(1) and with/without 50 microm PD98059. The collagen gels were released after 4 days and the percent contraction was determined by area measurements using image analysis. Differences in alpha-smooth muscle actin (alpha-SMA) and ERK-1/2 MAPK (phosphorylated and total) protein levels were analyzed with western blotting. RESULTS: Gels seeded with CVI fibroblasts contracted more than HS68, NC and LT fibroblasts. Inhibition of MAPK and/or stimulation with TGF-beta(1) increased the contraction of LC gels compared to unstimulated controls. Agonist induced gel contraction correlated with CVI disease severity. alpha-SMA protein expression in LC fibroblasts increased with MAPK inhibition with/without TGF-beta(1) stimulation, and correlated with the degree of gel contraction. Transfection with pCMV-Ras (activator of ERK-1/2) inhibited gel contraction; this inhibition was not reversed by addition of TGF-beta(1). Transfection with the pCMV-beta empty vector had no effect on gel contraction. CONCLUSIONS: TGF-beta1 stimulation of CVI patient fibroblasts grown in 3D collagen gels results in conversion to a contractile phenotype through upregulation of alpha-SMA, and in enhanced gel contraction. Inhibition of MAPK further increases gel contraction, while Ras activation of ERK-1/2 inhibits TGF-beta1-induced gel contraction. These responses correlate with increasing CEAP severity. CVI fibroblast mediated gel contraction is therefore regulated through cross-talk between the ERK-1/2 MAPK and TGF-beta(1) signalling cascades. These data identify potentially clinically relevant therapeutic molecular targets that could enhance matrix contraction and thereby improve venous ulcer wound healing.

Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching.

The minus strand and ambisense segmented RNA viruses include multiple important human pathogens and are divided into three families, the Orthomyxoviridae, the Bunyaviridae, and the Arenaviridae. These viruses all initiate viral transcription through the process of "cap-snatching," which involves the acquisition of capped 5' oligonucleotides from cellular mRNA. Hantaviruses are emerging pathogenic viruses of the Bunyaviridae family that replicate in the cytoplasm of infected cells. Cellular mRNAs can be actively translated in polysomes or physically sequestered in cytoplasmic processing bodies (P bodies) where they are degraded or stored for subsequent translation. Here we show that the hantavirus nucleocapsid protein binds with high affinity to the 5' cap of cellular mRNAs, protecting the 5' cap from degradation. We also show that the hantavirus nucleocapsid protein accumulates in P bodies, where it sequesters protected 5' caps. P bodies then serve as a pool of primers during the initiation of viral mRNA synthesis by the viral polymerase. We propose that minus strand segmented viruses replicating in the cytoplasm have co-opted the normal degradation machinery of P bodies for storage of cellular caps. Our data also indicate that modification of the cap-snatching model is warranted to include a role for the nucleocapsid protein in cap acquisition and storage.

Hantavirus N protein exhibits genus-specific recognition of the viral RNA panhandle.

A key genomic characteristic that helps define Hantavirus as a genus of the family Bunyaviridae is the presence of distinctive terminal complementary nucleotides that promote the folding of the viral genomic segments into "panhandle" hairpin structures. The hantavirus nucleocapsid protein (N protein), which is encoded by the smallest of the three negative-sense genomic RNA segments, undergoes in vivo and in vitro trimerization. Trimeric hantavirus N protein specifically recognizes the panhandle structure formed by complementary base sequence of 5' and 3' ends of viral genomic RNA. N protein trimers from the Andes, Puumala, Prospect Hill, Seoul, and Sin Nombre viruses recognize their individual homologous panhandles as well as other hantavirus panhandles with high affinity. In contrast, these hantavirus N proteins bind with markedly reduced affinity to the panhandles from the genera Bunyavirus, Tospovirus, and Phlebovirus or Nairovirus. Interactions between most hantavirus N and heterologous hantavirus viral RNA panhandles are mediated by the nine terminal conserved nucleotides of the panhandle, whereas Sin Nombre virus N requires the first 23 nucleotides for high-affinity binding. Trimeric hantavirus N complexes undergo a prominent conformational change while interacting with panhandles from members of the genus Hantavirus but not while interacting with panhandles from viruses of other genera of the family Bunyaviridae. These data indicate that high-affinity interactions between trimeric N and hantavirus panhandles are conserved within the genus Hantavirus.

Quasielastic 3He(e,e'p)2H reaction at Q2 = 1.5 GeV2 for recoil momenta up to 1 GeV/c.

We have studied the quasielastic 3He(e,e(')p)2H reaction in perpendicular coplanar kinematics, with the energy and the momentum transferred by the electron fixed at 840 MeV and 1502 MeV/c, respectively. The 3He(e,e(')p)2H cross section was measured for missing momenta up to 1000 MeV/c, while the A(TL) asymmetry was extracted for missing momenta up to 660 MeV/c. For missing momenta up to 150 MeV/c, the cross section is described by variational calculations using modern 3He wave functions. For missing momenta from 150 to 750 MeV/c, strong final-state interaction effects are observed. Near 1000 MeV/c, the experimental cross section is more than an order of magnitude larger than predicted by available theories. The A(TL) asymmetry displays characteristic features of broken factorization with a structure that is similar to that generated by available models.

Measurement of the 3He(e,e'p)pn reaction at high missing energies and momenta.

Results of the Jefferson Lab Hall A quasielastic 3He(e,e'p)pn measurements are presented. These measurements were performed at fixed transferred momentum and energy, q=1502 MeV/c and omega=840 MeV, respectively, for missing momenta p(m) up to 1 GeV/c and missing energies in the continuum region, up to pion threshold; this kinematic coverage is much more extensive than that of any previous experiment. The cross section data are presented along with the effective momentum density distribution and compared to theoretical models.

Role of matrix metalloproteinases 1, 2, and 9 and tissue inhibitor of matrix metalloproteinase-1 in chronic venous insufficiency.

PURPOSE: Increased transforming growth factor-beta(1) (TGF-beta(1)) activity is associated with chronic venous insufficiency (CVI) disease progression and dermal skin pathology. Because TGF-beta(1) stimulates collagen synthesis and alters the levels of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), we investigated the hypothesis that increased TGF-beta(1) activity is associated with differences in messenger RNA and protein levels of MMPs and TIMP-1 in patients with CVI. METHODS: One hundred ten biopsies of the lower calf and lower thigh in 73 patients were snap frozen in liquid nitrogen and stratified into six groups according to the clinical etiologic anatomic distribution pathophysiology disease classification. One set of lower-calf and lower-thigh biopsies were analyzed for MMP-1 and TIMP-1 gene expression with quantitative reverse transcription and competitive polymerase chain reaction. A second set of biopsies was analyzed for the active and latent forms of MMP-1, MMP-2, and MMP-9 as well as for TIMP-1 by western blotting, gelatin zymography, and tissue localization by immunohistochemistry (IHC). RESULTS: Compared with the control, MMP-1 messenger RNA was increased in class-4 and class-6 patients (P < or =.01), whereas TIMP-1 was increased in class-6 patients only (P < or =.05). However, there were no differences in total protein between MMP-1 and TIMP-1. Active MMP-2 protein increased in class-4 and class-5 patients compared with active MMP-1 and TIMP-1 (P < or =.01). Western blotting did not identify the active component of MMP-9. Similarly, only the latent form of MMP-9 was observed by gelatin zymography, whereas both the latent and active forms of MMP-2 were observed. IHC demonstrated MMP-1 and MMP-2 in dermal fibroblasts and in perivascular leukocytes. TIMP-1 was observed in basal-layer keratinocytes of the epidermis only. MMP-9 was not detected by IHC. CONCLUSION: MMP synthesis is regulated at both the transcriptional and post-transcriptional levels in CVI. Our data suggest that post-translational modifications are key to functional regulation. Dermal fibroblasts and migrating leukocytes are probable cellular sources of MMPs. Increased active MMP-2 levels in class-4 and class-5 patients indicate tissue remodeling caused by pre-ulcer and postulcer environmental stimuli. These data suggest that alterations in MMP-2 activity, in conjunction with TGF-beta(1)-mediated events, cause an imbalance in tissue remodeling leading to a pro-ulcer-forming environment.

VEGF increases permeability of the endothelial cell monolayer by activation of PKB/akt, endothelial nitric-oxide synthase, and MAP kinase pathways.

VEGF is a key regulator of vascular permeability. However, its signaling pathways are incompletely understood. We tested the hypothesis that VEGF regulates endothelial cell (EC) permeability by activating PKB/akt, NOS, and MAP kinase dependent pathways using human umbilical vein EC (HUVEC). Permeability was measured from FITC-dextran 70-kDa flux across the EC monolayer at baseline and after VEGF at 0.034, 0.068, 1, 10, and 100 nM. VEGF increased HUVEC permeability to FITC-dextran in a dose-dependent manner. VEGF (1 nM) increased permeability from 3.9 x 10(-6) +/- 0.7 x 10(-6) to 14.0 x 10(-6) +/- 1.7 x 10(-6) cm/s (mean +/- SEM; P < 0.001). Permeability changes were also assessed after treatment with 1, 10, and 100 nM wortmannin (PI 3-kinase inhibitor); 0.01, 0.1, and 1.0 nM LY294002 (PI 3-kinase inhibitor); 200 microM l-NMMA (NOS inhibitor); 2.7 microM AG126 (p42/44(MAPK) inhibitor); and 0.006, 0.06, and 0.6 microM SB203580 (p38(MAPK) inhibitor). All inhibitors blocked VEGF-induced permeability changes. Our data demonstrate that (1) VEGF increases permeability of EC monolayers in a dose-dependent fashion, and (2) VEGF-induced permeability is mediated through PI-3 kinase-PKB, NOS, and MAP-kinase signaling cascades. These observations suggest that microvascular hyperpermeability associated with inflammation and vascular disease is mediated by activation of these EC signaling pathways.

Repair bias of large loop mismatches during recombination in mammalian cells depends on loop length and structure.

Repair of loop mismatches was investigated in wild-type and mismatch binding-defective Chinese hamster ovary (CHO) cells. Loop mismatches were formed in vivo during extrachromosomal recombination between heteroallelic plasmid substrates. Recombination was expected to occur primarily by single-strand annealing (SSA), yielding 12- or 26-base nonpalindromic loop mismatches, and 12-, 26-, or 40-base palindromic loop mismatches. Nonpalindromic loops were repaired efficiently and with bias toward loop loss. In contrast, the 12-base palindromic loop was repaired with bias toward loop retention, indicating that repair bias depends on loop structure. Among the palindromic loops, repair bias was dependent on loop length, with bias shifting from loop retention to loop loss with increasing loop size. For both palindromic and nonpalindromic loops, repair efficiencies and biases were independent of the general (MSH/MLH) mismatch repair pathway. These results are discussed with respect to the maintenance of large nonpalindromic insertions, and of small and large palindromes, in eukaryotic genomes.

Stimulation of NO production and of eNOS phosphorylation in the microcirculation in vivo.

The role of nitric oxide (NO) in microvascular permeability is controversial, in part because the regulation of its endothelial constitutive synthase, eNOS, has been studied in vitro but not in vivo. Our study was designed to detect the morphologic and functional presence of eNOS and to test whether eNOS could be phosphorylated by platelet-activating factor (PAF), an agent that induces hyperpermeability. Immunocytochemistry was applied using human anti-eNOS antibodies in the hamster cheek pouch (hcp). The hcp microvessels demonstrated positive reaction products in the endothelium. The functional presence of eNOS in hcp was investigated by topical application of 10(-7) M PAF to the hcp and by measuring NO production by chemiluminescence. The mean baseline value of NO release was 63.3 +/- 6.9 pmol/ml (mean +/- SE). Application of PAF led to an increase in mean NO release to 120.8 +/- 31.2 pmol/ml (P < 0.05). In another series of experiments, 10(-7) M PAF was applied topically to hcp preincubated with [(32)P]orthophosphoric acid. Immunoprecipitation and Western blots detected (32)P-labeled bands that migrated with the mobility of positive eNOS indicating phosphorylated eNOS protein. The intensity of the radioactive bands was evaluated by computer-assisted image analysis. Comparison of the net band intensities yielded a mean PAF-treated/control ratio of 1.6 +/- 0.1. Our data demonstrate the morphologic and functional presence of eNOS in the microcirculation. The data also provide evidence that the function of microvascular eNOS is subject to regulation by phosphorylation.

Endotoxin-induced mesenteric microvascular changes involve iNOS-derived nitric oxide: results from a study using iNOS knock out mice.

The administration of endotoxin alters intestinal blood flow, increases nitric oxide (NO) production, and induces gut barrier dysfunction. Thus, we investigated the hypothesis that microvascular reactivity and permeability of the mesenteric vascular bed are altered to a lesser degree in iNOS knock out (iNOS -/-) mice than their wild-type (iNOS +/+) litter mates after an endotoxin challenge. To test this hypothesis, we compared the microvascular response of iNOS knockout (iNOS -/-) mice after a topical or systemic endotoxin challenge against that of their wild-type litter mates (iNOS +/+). Intravital microscopy was used to measure arteriolar diameter and postcapillary venular permeability in the mouse ileum. Both parameters were determined by computer-assisted image analysis. Diameter was measured in A1, A2, and A3 arterioles (1, 2, 3 = rank of deployment). Changes in microvascular permeability were measured from changes in interstitial fluorescence caused by extravasation of fluorescein isothiocyanate (FITC)-dextran 150 (molecular weight = 150 kDa) and expressed as changes in integrated optical intensity (IOI). In the first series of experiments, endotoxin (100 microg/mL) was applied topically to the ileal segment. In the second series, endotoxin (10 mg/kg) was administered intraperitoneally (i.p.). Administration of topical or i.p.. endotoxin caused vasoconstriction and was associated with an early increase in permeability in both iNOS +/+ and -/- mice, although over time the responses of the iNOS -/- and iNOS +/+ mice diverged. Twenty minutes after topical endotoxin, the increase in permeability in iNOS -/- mice had reached a plateau whereas it continued to increase in the iNOS +/+ mice, such that at 80 min post-topical endotoxin, IOI was 27+/-7 in iNOS -/- vs. 39+/-5 in iNOS +/+ (P < 0.05). A similar permeability response was observed after i.p.. endotoxin, where the increase in post-capillary venular permeability was greater in the iNOS +/+ mice (P < 0.05). Both iNOS -/- and iNOS +/+ mice had a similar transient vasoconstrictive response after topical endotoxin challenge (reduction in A2 arteriolar diameters by -17+/-4% vs. -24+/-7%), with return to baseline values by 60-80 min post-endotoxin challenge. The iNOS +/+ but not the iNOS -/- mice manifested a secondary vasodilatory response that persisted throughout the experimental period. The arteriolar vasoreactive response of the iNOS -/- and iNOS +/+ mice to i.p.. endotoxin was similar to that of topical endotoxin, but of a lesser magnitude. In conclusion, the similarity in effects between topical and systemic endotoxin indicates that endotoxin causes microvascular dysfunction in the gut by directly on the microcirculation. In addition, our data suggest that NO production by iNOS is involved in the microvascular alterations associated with gut barrier dysfunction.

Platelet-activating factor modulates microvascular dynamics through phospholipase C in the hamster cheek pouch.

We studied the interactions between platelet-activating factor (PAF) and phospholipase C (PLC) in the modulation of microvascular responses in the hamster cheek pouch using intravital microscopy and computer-assisted image analysis. Changes in arteriolar diameter and in integrated optical intensity (IOI, an index of vascular permeability) were measured. Fluorescein-isothiocyanate-labeled dextran 150 (FITC-Dx 150) served as a tracer for macromolecular transport. 2-Nitro-4-carboxyphenyl N,N-diphenylcarbamate (NCDC) and 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5,-dione (U-73122), two PLC inhibitors, were applied topically in separate experiments. PAF at 10(-7) M elevated IOI from baseline to a mean +/- SEM value of 70. 7 +/- 8.9 units. Pretreatment with 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated the maximal increment in mean IOI (+/-SEM) induced by PAF at 10(-7) M to mean +/- SEM values of 30.6 +/- 6.5, 39.3 +/- 6.0, 12.1 +/- 4.8, and 41.5 +/- 6.0, respectively. The simultaneous vasoconstrictor action of 10(-7) M PAF was expressed as the experimental-to-baseline ratio, with the baseline diameter adjusted to a value of 1. PAF constricted the arterioles to a mean +/- SEM ratio of 0.30 +/- 0.07. Pretreatment with the PLC inhibitors NCDC at 10(-4) and 10(-5) M NCDC and with U-73122 at 10(-5) and 10(-6) M attenuated 10(-7) M PAF-induced vasoconstriction to mean +/- SEM diameter ratios of 0.55 +/- 0.05, 0. 48 +/- 0.06, 0.55 +/- 0.08, and 0.58 +/- 0.06, respectively. Our results demonstrate that PLC is an element of the biochemical pathway involved in PAF modulation of microvascular permeability and in PAF modulation of arteriolar diameter.

Vein interposition cuffs decrease the intimal hyperplastic response of polytetrafluoroethylene bypass grafts.

PURPOSE: The modification of the distal anastomosis of polytetrafluoroethylene (PTFE) bypass grafts with vein interposition cuffs (VCs) has been reported to increase graft patency. However, the mechanisms that are responsible for this improved patency are unclear. Because intimal hyperplasia (IH) is a primary cause of prosthetic graft failure, we hypothesized that VCs affect the distal anastomosis by decreasing the IH response of the outflow artery. METHODS: Twenty-three female domestic Yorkshire pigs (mean weight, 35 kg) underwent 42 femoral PTFE bypass grafting procedures. The PTFE bypass grafts were separated into the following three groups according to distal anastomotic configuration: end-to-side anastomoses (ES), VCs, and cuffs constructed with PTFE (PCs). Four femoral arteries from two pigs served as healthy controls. At sacrifice, the grafts were perfusion fixed, and the distal anastomoses harvested at 1 and 4 weeks. The specimens were hemisected and serially sectioned to identify the heel, toe, and mid-anastomotic regions. The sections were cut into 5-microm segments and analyzed for intima and media thickness and area, intima/media area ratio, and the distribution of IH in the vein cuff. The roles of transforming growth factor-beta1 and platelet-derived growth factor-BB in IH development were assessed with immunohistochemistry. RESULTS: IH development was significantly lower at all areas of the anastomosis, with VCs compared with ES and PCs at 4 weeks (P

Adenoviral-mediated expression of antisense RNA to basic fibroblast growth factor reduces tangential stress in arterialized vein grafts.

PURPOSE: The purpose of this study was to test whether basic fibroblast growth factor (bFGF) participates in arterialized vein graft remodeling. METHODS: Rabbits underwent in vivo gene transfer and carotid interposition vein grafting. Segments of external jugular vein were infected with an adenovirus that expressed antisense bFGF RNA (Ad.ASbFGF) at 1 x 10(10) PFU/mL to inhibit new synthesis of bFGF by cells in the vein graft wall. Control rabbits were treated with either adenovirus that encoded beta-galactosidase (Ad.lacZ) at 1 x 10(10) PFU/mL or vehicle (phosphate-buffered saline solution [PBS]). At 3 days, 3 grafts per treatment group were harvested for the determination of gene expression of ASbFGF RNA by reverse transcriptase-polymerase chain reaction. Rabbits were killed, and perfusion was fixed 2 months after the grafting. Total wall thickness and lumen circumference of vein grafts and normal arteries were measured in cross sections. Calculated mean tangential stress (+/-SD) for the ASbFGF-treated group and controls was compared for significance. Grafts were immunohistochemically stained to assess bFGF protein production. RESULTS: Only the grafts infected with the Ad.ASbFGF gene expressed ASbFGF RNA. Grafts that were treated with Ad.ASbFGF displayed lower tangential stress (10.9 +/- 2.3 dynes/cm(2)) than PBS alone (22 +/- 2.8 dynes/cm(2)) or Ad. lacZ-treated controls (20.6 +/- 5.4 dynes/cm(2); P <.001). Tangential stress in the Ad.ASbFGF group was comparable to a normal carotid artery (13.9 +/- 2.1 dynes/cm(2)). The difference in mean total wall thickness was significant among the 3 treatment groups: Ad.ASbFGF, 164 +/- 3.4 microm); Ad.lacZ, 100 +/- 3.3 microm; and PBS, 96 +/- 3.6 microm; P <.01). Luminal circumference was not different among the groups. The Ad.ASbFGF-treated vein graft wall was composed of thick layers of concentric smooth muscle cells and elastin fibers in contrast to the sponge-like appearance observed in control arterialized vein grafts. Reduction in bFGF protein was noted only in the Ad.ASbFGF-treated group. CONCLUSION: Inhibition of bFGF synthesis in vivo with the use of adenoviral gene transfer of antisense RNA to bFGF promotes a vein graft with decreased tangential stress while maintaining the luminal area. The vein graft wall is remodeled and qualitatively resembles an artery so that wall tangential stress in Ad.ASbFGF and normal artery are not significantly different. The lack of significant difference in lumen circumference among groups suggests that wall thickening in the Ad. ASbFGF grafts is not at the expense of luminal narrowing. Our results suggest that ASbFGF RNA expression may represent an effective strategy in limiting the failure of arterialized venous conduits.

Microcirculatory inflammation in chronic venous insufficiency: current status and future directions.

One of the hallmarks of chronic venous insufficiency (CVI) is an elevated venous pressure. However, no direct link at the cellular and molecular levels between venous hypertension and actual tissue damage in CVI has been established. Evidence for generations of an inflammatory reaction and several molecular alterations in the development of CVI is discussed. Development of experimental models of CVI, analysis of the mechanical tissue stresses in addition to venous hypertension, in combination with systematic studies of clinically stratified and standardized patient-derived samples is required for molecular description of the pathogenesis of CVI.

Dermal tissue fibrosis in patients with chronic venous insufficiency is associated with increased transforming growth factor-beta1 gene expression and protein production.

PURPOSE: Pathologic dermal degeneration in patients with chronic venous insufficiency (CVI) is characterized by aberrant tissue remodeling that results in stasis dermatitis, tissue fibrosis, and ulcer formation. The cytochemical processes that regulate these events are unclear. Because transforming growth factor-beta(1) (TGF-beta(1)) is a known fibrogenic cytokine, we hypothesized that the increased production of TGF-beta(1) would be associated with CVI disease progression. METHODS: Seventy-eight punch biopsy specimens of the lower calf (LC) and the lower thigh (LT) of 52 patients were snap frozen in liquid nitrogen and stratified into four groups according to the Society for Vascular Surgery/International Society for Cardiovascular Surgery CEAP classification (C, clinical; E, etiologic; A, anatomic distribution; and P, pathophysiology). One set of LC biopsy specimens were analyzed for TGF-beta(1) gene expression with quantitative reverse transcriptase-polymerase chain reaction: healthy skin, n = 6; class 4, n = 6; class 5, n = 5; and class 6, n = 7. A second set of biopsy specimens from the LC and LT were analyzed for the amount of bioactive TGF-beta(1) with a certified cell line 64 mink lung epithelial bioassay: healthy skin, n = 8; class 4, n = 23; class 5, n = 13; and class 6, n = 10. The location of TGF-beta(1) was determined at the light and electron microscopy level with immunocytochemistry and immunogold (IMG) labeling. Multiple comparisons were analyzed with a one-way analysis of variance and the Student-Newman-Keuls post hoc tests. The LC and LT comparisons were analyzed with a two-tailed unpaired t test. RESULTS: The TGF-beta(1) gene transcripts for control subjects and patients in classes 4, 5, and 6 were 7.02 +/- 7.33, 43.33 +/- 9.0, 16.13 +/- 7.67, and 7.22 +/- 0.56 x 10(-14) mol/microg total RNA, respectively. The transcripts were significantly elevated in class 4 patients only (P

Hemodialysis access: influence of the human immunodeficiency virus on patency and infection rates.

PURPOSE: The complication rate for patients who are dialysis dependent and infected with the human immunodeficiency virus (HIV) and the role of viral indicators (CD4 counts) as predictors of these complications are poorly characterized. To determine the influence of HIV status and viral activity on graft patency and infection rates, we retrospectively reviewed our results. METHODS: Between June 1993 and March 1997, the charts of 104 patients (HIV+, n = 42; HIV-, n = 62) who required 112 hemodialysis access grafts were reviewed. Of the 112 procedures, 55 (48%) were autologous arteriovenous fistulae (AVF) procedures (HIV+, n = 23; HIV-, n = 32) and 57 (52%) were prosthetic expanded polytetrafluoroethylene grafting procedures (HIV+, n = 27; HIV-, n = 30). Transcutaneous catheter procedures were excluded from the study. The autologous AVF procedures consisted of direct and transposed AVFs. Patency rates were determined by means of life-table analysis. Infection rates and CD4 counts were compared with the chi2 test and the Fisher exact test. Significance was accepted at a P value of.05 or less. RESULTS: The cumulative 12-month and 24-month patency rates for prosthetic grafts in patients who were HIV+ were 49% and 21%, respectively, versus 77% and 45% for patients who were HIV-. The differences in the prosthetic graft patency rates between these two groups were significant (P .05). The mean CD4+ cell counts were 174: CD4+ counts that were less than 200 did not correlate with or predict the development of infection (P >.05). CONCLUSION: Our data showed that prosthetic graft infection rates were increased and patency rates were decreased in patients who were HIV+ as compared with patients who were HIV- and HIV+ with autologous AVFs. There were no differences in patency rates or infection rates in patients who had undergone autologous access procedures. Long-term graft patency rates were not affected by HIV status, and CD4+ lymphocyte counts were not predictive of infection development. Because the prosthetic graft infection rates exceeded those rates of autologous access procedures, we recommend the vigorous use of autologous AVFs in all patients who are HIV+, regardless of CD4+ count.

Microvascular pathophysiology of skeletal muscle ischemia-reperfusion.

Ischemia-reperfusion injury is a continuing challenge to vascular surgeons. Microvascular factors and mechanisms underlying edema and compartment syndrome provide a useful end point for analysis and planning of therapy. We review the pivotal role of endothelium-leukocyte interactions and of cytokines in the genesis of postischemic damage in muscle. Clinical considerations and future directions based on research and practice are presented.

Retinoblastoma protein: a molecular regulator of chronic venous insufficiency.

PURPOSE: Chronic venous insufficiency (CVI) and varicose vein (VV) formation is characterized histologically by the transformation of smooth muscle cells (SMC) from a contractile to a secretory phenotype and by intense collagen deposition. The subcellular regulation point for these processes may be the retinoblastoma protein (pRb), a known inhibitor of cellular proliferation and regulator of differentiation. We hypothesize that pRb phosphorylation is associated with VV formation and functions as a possible subcellular regulator. METHODS: Patients were separated into two groups. Group 1 (n = 6) consisted of vein specimens obtained from patients undergoing coronary artery bypass grafting. Group 2 (n = 6) consisted of patients with symptomatic CVI and duplex confirmed refluxing greater saphenous veins (GSVs) who required GSV stripping. Western blots of GSV protein extracts were performed with anti-human pRb monoclonal antibodies and the degree of nonphosphorylated and phosphorylated pRb was determined. Results were quantified using image analysis of band intensities (computer calibrated intensity units). The ultrastructural appearance of SMCs and the vein wall architecture were qualitatively analyzed with electron microscopy in both groups. RESULTS: Phosphorylated pRb from varicose GSVs exhibited intensities of 523 +/- 188 units, while phosphorylated pRb from normal GSVs demonstrated intensities of 153 +/- 41 units (P < 0.05). SMCs in varicosed GSVs were surrounded by disorganized collagen deposits and displayed a secretory phenotype with spherical vacuolated cells. SMCs from normal GSVs appeared spindle shaped with a purported contractile phenotype and a well-structured extracellular matrix. CONCLUSION: Our data demonstrate that VV formation, in patients with CVI, is associated with phosphorylated pRb and the transformation of SMCs from a contractile to a secretory ultrastructural morphology. The data suggest that SMC dedifferentiation is regulated by pRb and the disinhibition of this protein (phosphorylation) may be an significant factor in the development of lower extremity varicosities.