In vitro measurements of hemodynamic forces and their effects on endothelial cell mechanics at the sub-cellular level.

This paper presents micro-particle tracking velocimetry measurements over cultured bovine aortic endothelial cell monolayers in microchannels. The objective was to quantify fluid forces and cell morphology at the sub-cellular scale for monolayers subjected to steady shear rates of 5, 10, and 20 dyn/cm2. The ultimate goal of this study was to develop an experimental methodology for in vitro detailed study of physiologically realistic healthy and diseased conditions. Cell topography, shear stress, and pressure distributions were calculated from sets of velocity fields made in planes parallel to the microchannel wall. For each experiment, measurements were made in 3 h intervals for 18 h. It was found that there is a three-dimensional change in cell morphology as a result of applied shear stress. That is, cells flatten and become more wedge shaped in the stream direction while conserving volume by spreading laterally, i.e., in the cross-stream direction. These changes in cell morphology are directly related to local variations in fluid loading, i.e., shear stress and pressure. This paper describes the first flow measurements over a confluent layer of endothelial cells that are spatially resolved at the sub-cellular scale with a simultaneous temporal resolution to quantify the response of cells to fluid loading.

Xenogeneic skin graft rejection in M-CSF/macrophage deficient osteopetrotic mice.

BACKGROUND: The cellular infiltrate in xenografts suggests that macrophages may be involved in xenograft rejection. However, the precise role of macrophages in xenograft rejection has not yet been fully addressed. METHODS: Xenogeneic rat skin grafts were transplanted to macrophage colony stimulating factor (M-CSF)/macrophage-deficient osteopetrotic ([OP]-/-) and wild-type control mice. Skin graft survival and antidonor rat humoral responses were quantified. RESULTS: Xenogeneic rat skin grafts survived 13 days in wild-type control mice, survival of rat skin grafts was significantly prolonged to 24 days in [OP]-/- mice (P<0.01). Similar results were observed in sensitized [OP]-/- and control mouse recipients, showing markedly prolonged rat skin graft survival in [OP]-/- mice. Levels of T-cell-dependent antirat antibodies [immunoglobulin G (IgG)2a and IgG3] in sera of [OP]-/- mice were significantly lower than that of control mice 2 weeks post-rat skin grafting. The proliferative responses to xenogeneic rats not to allogeneic mouse stimulation of T cells from [OP]-/- mice were significantly lower than that of wild-type mice. However, neutrilization of M-CSF by anti-M-CSF monoclonal antibody (mAb) or the addition of M-CSF to the in vitro culture systems of wild-type or [OP]-/- mouse T-responder cells, respectively, did not significantly change proliferative responses and cytolytic function against xenogeneic rat targets of wild-type or [OP]-/- mouse T-responder cells. CONCLUSIONS: The in vitro data indicate that M-CSF does not directly regulate cellular immune responses to xenoantigens. The present studies indicate that macrophages may play an important role in immune rejection of xenografts. The precise role of macrophages in xenograft rejection should be further investigated.

Angioplasty with stenting is effective in treating blue toe syndrome.

Blue toe syndrome is a manifestation of distal embolization associated with significant pain and risk of tissue loss. The recommended treatment options for this problem include endarterectomy or bypass with exclusion of the source of emboli. Although focal arterial stenosis can be effectively treated with angioplasty,it is unclear whether performing angioplasty in a lesion suspected of causing distal embolization might actually worsen the condition or what long-term effects this would have in preventing future embolization. The purpose of this study was to evaluate the treatment and outcome of a series of patients with unilateral blue toe syndrome treated with percutaneous angioplasty and stenting. During a 5-year period, a total of 8 patients were identified with unilateral blue toe syndrome. Ankle/brachial indices (ABIs) were obtained, followed by arteriography. The study group included 4 men and 4 women with an age range of 35 to 83 years. Their atherosclerotic risk factors included smoking (8), hypertension (5), diabetes mellitus (3), and hypercholesterolemia (1). One patient had a history of illicit drug use. The patients were followed up by repeat clinical examinations and vascular laboratory studies. Arteriography typically demonstrated a focal preocclusive lesion with thrombus at the distal end of the lesion. Angioplasty and stent placement was technically successful in all cases. The ABIs increased following angioplasty (before 0.81 +/- 0.05; after 1.02 +/-.05). The symptoms resolved in all 8 patients over the ensuing month, and there were no recurrences with a mean follow-up of 18.5 months (range 4 to 36 months). There was 1 death at 4 months associated with preexisting colon carcinoma. Unilateral arterial to arterial emboli were found in association with focal preocclusive lesions. Despite the presence of thrombus in some of the lesions, these patients were not acutely worse following angioplasty. There was good initial angiographic success in all cases. There was also hemodynamic improvement as shown by the increased ankle/brachial indices. Although long-term follow-up is not available, these intermediate results suggest that angioplasty and stenting should be considered a reasonable alternative to standard operative approaches for patients with blue to syndrome associated with embolization from a focal stenosis.

Cytokine pattern in aneurysmal and occlusive disease of the aorta.

BACKGROUND: Prominent inflammatory infiltrates of macrophages and T-lymphocytes are found in both aortic occlusive disease (AOD) and abdominal aortic aneurysms (AAA). These cells secrete different cytokines that might affect matrix turnover through modulation of matrix metalloproteinase expression. A different cytokine pattern might account for the evolution of AOD vs AAA. MATERIALS AND METHODS: Six different cytokines were examined to determine whether AOD and AAA could be characterized by unique cytokine patterns. AOD (n = 8) and AAA (n = 8) tissues were collected and serially treated with salt, dimethyl sulfoxide, and urea buffers to extract the soluble matrix or cell-bound cytokines. Levels of IL-1 beta, TNF-alpha, IL-10, IL-12, and IFN-gamma were measured by immunoenzymatic methods. Additionally, RNA levels of IL-12 and IFN-gamma were measured. RESULTS: AAA tissue contained higher levels of IL-10 compared to AOD tissue (P < 0.05). Higher levels of the proinflammatory cytokines IL-1 beta, TNF-alpha, and IL-6 were found in AOD (P < 0.05). mRNA levels of IL-12 and IFN-gamma did not differ between the diseases. Aortic tissues contained large amounts of matrix or cell-bound cytokines. CONCLUSIONS: AAA is characterized by greater levels of IL-10 while IL-1 beta, TNF-alpha, and IL-6 are higher in AOD. Targeted deletion of these cytokines in animal models might help in identifying their role in the progression of AAA.

The expression and localization of membrane type-1 matrix metalloproteinase in human abdominal aortic aneurysms.

BACKGROUND AND OBJECTIVE: Matrix metalloproteinase-2 (MMP-2) degrades both fibrillar collagens and elastin. MMP-2 is secreted as a latent 72-kd proenzyme that must be proteolytically processed to the 62-kd active form. In our laboratory we demonstrated a significant increase of active, matrix-bound MMP-2 in abdominal aortic aneurysmal (AAA) tissue compared with nonaneurysmal aorta with arteriosclerotic occlusive disease and normal aortic tissue. This increase in active MMP-2 is considered to be important in aneurysm pathogenesis, but the mechanism of its activation in aortic tissue is unknown. Membrane type-1 MMP (MT-1 MMP) is known to be an activator of MMP-2. The purpose of this study was to determine MT-1 MMP expression and its involvement in pro-MMP-2 activation in human aneurysmal tissue. METHODS: Infrarenal aortic tissue was obtained during the surgical repair of AAAs or the bypass of aortoiliac occlusive disease, or from nondiseased aorta, and the expression of MT-1 MMP messenger RNA was determined with Northern blot analysis. MT-1 MMP protein was determined with immunoblot and immunohistochemistry. The ability of aortic tissue to activate pro-MMP-2 was analyzed by incubating aortic tissue with exogenous radiolabeled pro-MMP-2. RESULTS: MT-1 MMP messenger RNA and protein are increased in AAA (P <.05) compared with arteriosclerotic occlusive disease and normal aortic tissue. Immunohistochemical analysis localized MT-1 MMP to aortic smooth muscle cells and macrophages in aneurysmal tissue. AAA tissue demonstrated a greater capacity to activate exogenous pro-MMP-2 compared with atherosclerotic and normal aortic tissue (P <.05). CONCLUSION: These studies demonstrate that MT-1 MMP is increased in AAA tissue and suggest that it may be important in AAA pathogenesis through its ability to activate pro-MMP-2

Microbubble potentiated ultrasound as a method of declotting thrombosed dialysis grafts: experimental study in dogs.

Intravenous perfluorocarbon-exposed sonicated dextrose albumin (PESDA) microbubbles in the presence of low frequency ultrasound (LFUS) can lyse very small clots. We develop a similar method to declot full-size arteriovenous dialysis grafts. Dialysis grafts fashioned in three dogs were cannulated and ligated. After thrombosis, three declotting techniques were randomly applied: 1) direct injection of PESDA + LFUS; 2) direct injection of saline + LFUS; and 3) intravenous PESDA + LFUS. Declotting was graded by cine angiography scores of each third of the graft on a scale of 0-4 (maximum total score = 12). Twenty-six procedures showed mean patency scores of 11.1 for direct PESDA and 8.4 for i.v. PESDA, vs 4.9 for direct saline, p = <0.001. All eight direct PESDA injections achieved lysis and good flow, but none of 8 direct saline injections succeeded, p = <0.01. Intravenous PESDA succeeded in 4 of 10 procedures, p = <0.04 vs saline. Direct injection of PESDA with transcutaneous LFUS succeeds in lysing moderate-size clots and recanalizing thrombosed fistulas.

MMP inhibition in abdominal aortic aneurysms. Rationale for a prospective randomized clinical trial.

Abdominal aortic aneurysms (AAAs) represent a chronic degenerative condition associated with a life-threatening risk of rupture. The evolution of AAAs is thought to involve the progressive degradation of aortic wall elastin and collagen, and increased local production of several matrix metallo-proteinases (MMPs) has been implicated in this process. We have previously shown that tetracycline derivatives and other MMP inhibitors suppress aneurysm development in experimental animal models of AAA. Doxycycline also reduces the expression of MMP-2 and MMP-9 by human vascular wall cell types and by AAA tissue explants in vitro. To determine whether this strategy might have a role in the clinical management of small AAA, we examined the effect of doxycycline on aortic wall MMP expression in vivo. Patients were treated with doxycycline (100 mg p.o. bid) for 7 days prior to elective AAA repair, and aneurysm tissues were obtained at the time of surgery (n = 5). Tissues obtained from an equal number of untreated patients with AAA were used for comparison. By reverse transcription-polymerase chain reaction and Southern blot analysis, MMP-2 and MMP-9 were both found to be abundantly expressed in the aneurysm wall. Preoperative treatment with doxycycline was associated with a 3-fold reduction in aortic wall expression of MMP-2 and a 4-fold reduction in MMP-9 (p < 0.05 compared to untreated AAA). These preliminary results suggest that even short-term treatment with doxycycline can suppress MMP expression within human AAA tissues. Given its pleiotropic effects as an MMP inhibitor, doxycycline may be particularly effective in suppressing aortic wall connective tissue degradation. While it remains to be determined whether MMP inhibition will have a clinically significant impact on aneurysm expansion, it is expected that this question can be resolved by a properly designed prospective randomized clinical trial.

Doxycycline induces Fas/Fas ligand-mediated apoptosis in Jurkat T lymphocytes.

Tetracyclines have been used in the treatment of chronic inflammatory diseases associated with local infiltration of inflammatory cells and matrix destruction as observed in rheumatoid arthritis and periodontal disease. Fas/Fas ligand (FasL)-mediated apoptosis plays an important role in maintaining T lymphocyte homeostasis and modulating immune response. The present study demonstrates that doxycycline inhibits Jurkat T lymphocyte proliferation and induces apoptosis. The phytohemagglutinin (PHA)-activated Jurkat cells are more susceptible to doxycycline-induced apoptosis. Furthermore, doxycycline-induced apoptosis is associated with increased Fas/FasL expression in Jurkat cells. The increase of apoptosis in Jurkat cells treated with doxycycline is consistent with the increase of FasL expression. These results suggest that doxycycline may downregulate the inflammatory process in certain diseases by eliminating activated T lymphocytes through Fas/FasL-mediated apoptosis.

Association of malondialdehyde-acetaldehyde (MAA) adducted proteins with atherosclerotic-induced vascular inflammatory injury.

Atherosclerosis is a vascular injury characterized by elevated tissue levels of tumor necrosis factor-alpha (TNF-alpha), increased expression of endothelial cell adhesion molecules, and vascular wall inflammatory cell infiltration. Foam cells are associated with atherosclerotic plaque material, and low density lipoprotein (LDL) is a lipid component of foam cells. Malondialdehyde (MDA) is an oxidative product of unsaturated fatty acids and is also present in atherosclerotic lesions. MDA-modified (adducted) proteins, including MDA-modified LDL, are present in atherosclerotic human vascular tissue. Acetaldehyde (AA) is the major metabolic product of ethanol oxidation. Both MDA and AA are highly reactive aldehydes and will combine with proteins to produce an antigenically distinct protein adduct, termed the MAA adduct. This study demonstrates that proteins modified in the presence of high concentrations of MDA can produce MAA-modified proteins in vitro. In addition, MAA adducted proteins are capable of inducing rat heart endothelial cell cultures (rHEC) to produce and release TNF-alpha, and cause rHEC upregulation of endothelial adhesion molecule expression, including ICAM-1. These adhesion molecules are required for circulating inflammatory cells to adhere to endothelium which allows inflammatory cell tissue infiltration. Additionally, MAA modified proteins were defected in human atherosclerotic aortic vascular tissue but not in normal aortic tissue. Since atherosclerosis is associated with an inflammatory vascular injury characterized by elevated tissue TNF-alpha concentrations and inflammatory cell infiltration, these data suggest that MAA-adducted proteins may be formed in atherosclerotic plaque material and may be involved in the inflammatory reaction that occurs in atherosclerosis. These data further suggest that previous studies demonstrating MDA modified protein in atherosclerotic plaque may in fact have MAA modified proteins associated with them.

Distal radial artery lesion as a source of digital emboli.

Ischemic changes of the digits caused by emboli are rare. When they do occur, the typical sites of origin include the heart, the proximal subclavian artery, and the thoracic outlet. Dialysis access or iatrogenic injuries may be a more distal source of emboli. Two patients, each with embolization to the thumb and index finger from a lesion in the anatomical snuff-box, were studied. Neither patient had any other atherosclerotic occlusive disease, and both lesions occurred precisely where the extensor pollicis longus crossed the artery and would be expected to compress it against the proximal epiphysis of the first metacarpal when the hand was closed. These lesions were excised, and bypass was performed, with rapid resolution of symptoms. This is an unusual cause of digital embolization that should be considered in patients with emboli to the thumb and index finger.

The formation of aneurysms.

Arterial aneurysms account for a significant proportion of the various diseases treated by the vascular surgeon. Refinements of surgical technique have reduced the morbidity and mortality, yet, we have no effective medical therapy to prevent the growth of small aneurysms. Although the pathogenesis of aneurysmal disease has received attention, the complex nature of the process has not been fully elucidated. The emergence of new and refined techniques in the fields of immunology, biochemistry, cell biology, and genetics has advanced the understanding of the dynamic interactions within a diseased vessel. Although past work was descriptive, investigators are now studying the role of the local inflammatory infiltrates and the destructive proteolytic enzymes they produce and regulate. The clinical observations we make regarding the familial tendency of abdominal aortic aneurysms (AAA) underscores the importance of research directed at identifying an aneurysm-related gene. As new pieces are added to the puzzle and the picture of AAA pathogenesis becomes more clear, we can expect the development of new therapeutic measures directed at controlling the critical matrix changes, and thus the growth of small AAA, as well as screening methods searching for AAA-associated genes.

Matrix metalloproteinase-2 production and its binding to the matrix are increased in abdominal aortic aneurysms.

Degradation of the elastic media is a hallmark of abdominal aortic aneurysms (AAAs). We examined the expression of 2 elastolytic matrix metalloproteinases (MMPs), MMP-2 and MMP-9, in AAA aortic tissues compared with those from atherosclerotic occlusive disease (AOD) and nondiseased control tissues. Quantitative competitive reverse transcription-polymerase chain reaction and gelatin zymography showed increased MMP-9 mRNA and protein in both AAA and AOD tissues compared with those in control tissue, but there was no significant difference between AAA and AOD. In contrast, MMP-2 mRNA and protein levels were significantly higher in AAA than in AOD or control tissues. Sequential extraction of the MMPs from the aortic tissue with a physiological salt solution, 2% dimethylsulfoxide (DMSO), and 10 mol/L urea showed that large amounts of MMP-2 and MMP-9 were bound to the matrix. The most conspicuous finding was that the levels of MMP-2 were significantly elevated in the DMSO fraction in AAA tissues compared with AOD and control tissues. In addition, a large portion of MMP-2 found in the DMSO and urea fractions was in the active 62-kDa form, indicating that the precursor of MMP-2 in AAA is largely activated locally and binds to the tissue matrix tightly. By immunolocalization, MMP-9 was found to be primarily produced by macrophages and MMP-2 by mesenchymal cells. The production of MMP-2 was prominent when mesenchymal cells were surrounded by inflammatory cells, suggesting paracrine modulation of MMP-2 expression in AAAs. These observations emphasize that MMP-2 participates in the progression of AAAs by degrading aortic tissue matrix components.

Hospital-wide educational program decreases red blood cell transfusions.

BACKGROUND: Because of the numerous risks associated with the use of packed red blood cells (RBCs), it is critical that they be transfused only when appropriate. A hospital-wide educational program was developed in an attempt to improve the transfusion practices and provide a framework for blood bank audit at a Veterans Affairs teaching hospital. MATERIALS AND METHODS: The program required physicians to fill out an information sheet that listed appropriate criteria for transfusion. Charts were reviewed to determine if the transfusion met these criteria. If the transfusion was deemed inappropriate by peer review, the staff physician was notified by letter. The information sheet was used on a voluntary basis without chart review in 1989 and on a mandatory basis beginning in 1990. Transfusion rates and mortality were adjusted to patient days of hospitalization and evaluated using chi 2 analysis. RESULTS: While voluntary use did not affect transfusion rate, mandatory implementation resulted in a 26% decline (P < 0.001) between 1989 and 1990 in the number of RBC units transfused per patient days of hospitalization. A diminished use of RBCs persisted in the subsequent years. There was no increase in mortality during this time to suggest a detrimental effect from the decrease in RBC transfusion. No apparent variation in the hospital population could account for the changes. CONCLUSION: Use of a unique and simple transfusion request sheet as an educational tool resulted in improved transfusion practices at a Veteran Affairs teaching hospital.

Carotid string sign resulting from an aberrant branch of the internal carotid artery.

Branches of the extracranial internal carotid artery are very rare. A case is reported wherein an aberrant artery originated from the bulb of the internal carotid artery (ICA) approximately 2 cm from the bifurcation. The ICA was occluded distal to the branch's origin. Arteriography in this case gave the appearance of a carotid "string sign". Vascular surgeons and radiologists should be aware of this anomaly when interpreting carotid arteriograms.

Protein kinase C isoforms in human aortic smooth muscle cells.

PURPOSE: To identify the protein kinase C (PKC) isoforms in human arterial smooth muscle cells (SMC) and define their subcellular location in the resting state and in response to the PKC activator, 12-O-tetradecanoylphorbol 13-acetate (TPA). METHODS: Arterial SMC cultures established from transplant donor aorta were treated with 100 nM TPA or control media, then mechanically lysed. PKC from the soluble and particulate fraction were separated by centrifugation, and protein normalized immunoblots were performed with antibodies to the PKC isoforms alpha, betaI, betaII, delta, epsilon, gamma and zeta. Bands were detected by enhanced chemiluminescence and analyzed densitometrically, with results expressed as the mean percentage of each fraction +/- SEM. Translocation was defined as a significant (p < 0.05) change in the particulate fraction for each isoform. Immunofluorescent staining of cultured SMC visualized the resting location and stimulated translocation of each isoform. RESULTS: Isoforms alpha and betaI were detected primarily in the soluble fraction, translocating to the particulate fraction with TPA stimulation (p < 0.0001). The isoforms betaII, delta, and epsilon were found primarily in the particulate fraction and did not translocate. Immunofluorescent staining confirmed these locations. Neither gamma or zeta were detected in these SMC. CONCLUSIONS: The PKC isoforms expressed in human arterial SMC differ from those reported in animal models. Their specific locations and response to stimulation suggest unique functions in cellular regulation and provide the groundwork for further investigation into their role in the development of vascular disease and regulation of matrix metabolism.

Pathogenesis of abdominal aortic aneurysm: an update and look toward the future.

To date, aneurysm research has been primarily descriptive, reiterating the complex nature of the disease process. Enhanced by the convergence of matrix biochemistry, cell biology and immunology, this work is providing important new insight into how matrix metabolism is regulated in the diseased aorta. The focus is now on the inflammatory process and its regulation of the matrix remodeling which occurs with abdominal aortic aneurysm. A family of matrix-degrading enzymes appear to have a central role in this process. As we have learned from the evolution of the treatment of other pathologic processes such as peptic ulcer disease, the most effective pharmacologic therapies are designed from a thorough understanding of the pathophysiology of the disease. We are quickly moving forward in formulating a comprehensive understanding of the various complex interactions that result in the formation of aortic aneurysm. Given the progress of the past decade, we can expect the identification of aneurysm-associated genes and clinical trials of anti-inflammatory medications and protease inhibitors as we enter the 21st century.

Staple occlusion of the infrarenal aorta: a videoscopic retroperitoneal approach.

PURPOSE: The purposes of this study were to determine whether available laparoscopic stapling devices could be used to interrupt the diseased human aorta, and to develop a videoscopic technique for retroperitoneal exposure and control of the infrarenal aorta in pigs. Our long-term goal is to develop a minimally invasive approach to the treatment of abdominal aortic aneurysms by exclusion and extraanatomic bypass. METHODS: Ten diseased, formalin-preserved human cadaver aortas underwent stapling using a laparoscopic stapling device. The aortas were then pressurized to superphysiologic levels to assess the integrity of the staple line. Ten swine underwent retroperitoneal video-assisted exploration with control and staple occlusion of the aorta and iliac artery. RESULTS: The staple line was complete and remained intact after pressurization in nine of 10 cadaver aortas, despite the presence of complex calcified disease. One aorta had a 2-mm opening through the staple line. Through the left retroperitoneal approach, the infrarenal aorta and left iliac artery could be dissected and controlled. A modified pledgeted technique used for stapling resulted in hemostasis of the staple line and exclusion of flow without injury to adjacent structures. CONCLUSIONS: The diseased human aorta can be occluded using available laparoscopic staplers. These swine experiments demonstrate the feasibility of the retroperitoneal approach for exclusion of infrarenal aortic aneurysms.