Filgrastim, fibrinolysis, and neovascularization.

Segmental recanalization of chronically occluded arteries was observed in patients with chronic limb-threatening ischemia (CLTI) treated with Filgrastim, a granulocyte colony stimulating factor, every 72 h for up to a month, and an infra-geniculate programmed compression pump (PCP) for 3 h daily. Molecular evidence for fibrinolysis and neovascularization was sought. CLTI patients were treated with PCP alone (N = 19), or with Filgrastim and PCP (N = 8 and N = 6, at two institutions). Enzyme-Linked Immunosorbent Assay was used to measure the plasma concentration of plasmin and of fibrin degradation products (FDP), and the serum concentration of proteins associated with neovascularization. In the PCP-alone group, blood was sampled on Day 1 (baseline) and after 30 days of daily PCP. In the Filgrastim and PCP group, blood was drawn on Day 1, and 1 day after the 5th and the 10th Filgrastim doses. Each blood draw occurred before and after 2 h of supervised PCP. Significant (p < 0.01) PCP independent increases in the plasma concentration of plasmin (>10-fold) and FDP (>5-fold) were observed 1 day after both the 5th and the 10th Filgrastim doses, compared to Day 1. Significant (p < 0.05) increases in the concentration of pro-angiogenic proteins (e.g., HGF, MMP-9, VEGF A) were also observed. Filgrastim at this novel dosimetry induced fibrinolysis without causing acute hemorrhage, in addition to inducing a pro-angiogenic milieu conducive to NV. Further clinical testing is warranted at this novel dosimetry in CLTI, as well as in other chronically ischemic tissue beds. Trial registration. https://clinicaltrials.gov/ct2/show/NCT02802852.

Splenic artery as a conduit to facilitate visceral arterial reconstruction.

Splenic-to-superior mesenteric artery transposition was used to treat proximal celiac in-stent occlusion in one patient and to prepare a landing zone for thoracic endograft treatment of a dissection in another. The proximal splenic artery was used as a conduit to facilitate visceral aortic debranching in four patients. Using the splenic artery as a conduit to preserve or restore celiac perfusion without interrupting liver perfusion is feasible.

Development of Mega-Aorta Following Incompletely Treated Giant Cell Arteritis.

An 82-year-old male presented with a 9.3 cm ascending aorta and arch aneurysm with additional aneurysms of the innominate, right subclavian, and left common carotid arteries. The patient had a history of temporal arteritis that was only briefly treated in 1989 and a 6 cm ascending aortic aneurysm that was repaired in 1993. Our operative strategy was to construct a temporary parallel cerebrovascular circuit for cerebral protection during the redo-sternotomy and aortic arch reconstruction, with the added benefit of permanently excluding the branch arch vessel aneurysms. Pathological analysis of the aortic specimen at the first operation may have identified giant cell arteritis, prompting medical therapy against further disease progression.

Successful use of ECMO in a Jehovah's Witness after complicated re-heart transplant.

We present a 29-year-old male Jehovah's Witness, who underwent a second orthotopic heart transplantation 20 years after his first transplant. After extubation, the patient aspirated and required support with extracorporeal membrane oxygenation (ECMO). During the period of ECMO support, his hemoglobin decreased to 4.7 mg/dl. The patient was ultimately weaned from ECMO and discharged home. To our knowledge, this is the first reported case of 1) heart retransplantation and 2) rescue with ECMO in a Jehovah's Witness patient.

Contemporary outcomes in vascular patients who require preoperative coronary stent.

BACKGROUND: The documented risks of preoperative coronary revascularization prior to vascular surgery have led to a marked reduction in the role of percutaneous coronary intervention (PCI) during preoperative risk stratification. However, many patients with peripheral arterial disease are first identified immediately after a PCI for an acute coronary syndrome. We sought to determine the risks associated with these patients who then go on to have a peripheral arterial intervention (open operation or endovascular procedure). We hypothesized that there was no difference in outcomes in patients whose medical condition required PCI with coronary stent placement prior to a vascular operation compared with a control cohort of nonstented patients who underwent a vascular operation alone. We report the vascular operative outcomes in a contemporary cohort of vascular patients who had PCI with coronary stent placement for an acute event. METHODS: We performed a retrospective cohort analysis, utilizing administrative data, of 3,678 vascular patients from 2005 to 2010 at a tertiary care hospital. Two groups were defined: patients with preoperative PCI and coronary stent placement within 1 year prior to vascular operation (N = 101, mean age 66 ± 1.22 years, 51.5% men); and patients with no PCI prior to vascular operation (N = 3,577, mean age 60 ± 0.27 years, 46.37% men). Cardiovascular risk factors and complications derived from ICD-9 codes were used to parse data after open peripheral vascular surgery, endovascular repair, or amputation. Primary outcomes were death, nonfatal myocardial infarction, major adverse cardiac event (MACE, defined as death, myocardial infarction, or subsequent coronary revascularization) or bleeding. RESULTS: Univariate analysis showed significant differences in both demographic and outcome analysis in patients with and without prior coronary stent. Patients with a recent PCI followed by a vascular procedure were more likely to undergo an endovascular procedure (75.3% vs. 64.5%, odds ratio = 1.67, P = 0.028). These patients also had 11 of 20 cardiovascular risk factors, significantly higher than in those without a prior PCI. Multivariate subgroup analysis indicated that patients with a prior coronary stent were more likely to have an episode of congestive heart failure (CHF) after 1 year of surgery (16.8%, P = 0.045). In addition, an acute cardiac ischemic event was more likely within 1 year (2.0%, P = 0.036) and beyond 1 year (4.0%, P = 0.022) of surgery. Importantly, there was no significant increase in death, myocardial infarction, MACE, or bleeding in patients with a preoperative coronary stent. CONCLUSIONS: Patients who underwent PCI with coronary stent and then went on to require a vascular procedure had significantly more cardiovascular (CV) risk factors and were more likely to have an endovascular procedure than those patients without preoperative PCI. When controlling for CV risk factors and procedure type, there was no significant difference in death, MI, MACE, or bleeding complications between the groups.

Defective CXCR4 expression in aged bone marrow cells impairs vascular regeneration.

The chemokine stromal cell-derived factor-1 (SDF-1) plays a critical role in mobilizing precursor cells in the bone marrow and is essential for efficient vascular regeneration and repair. We recently reported that calcium augments the expression of chemokine receptor CXCR4 and enhances the angiogenic potential of bone marrow derived cells (BMCs). Neovascularization is impaired by aging therefore we suggested that aging may cause defects of CXCR4 expression and cellular responses to calcium. Indeed we found that both the basal and calcium-induced surface expression of CXCR4 on BMCs was significantly reduced in 25-month-old mice compared with 2-month-old mice. Reduced Ca-induced CXCR4 expression in BMC from aged mice was associated with defective calcium influx. Diminished CXCR4 surface expression in BMC from aged mice correlated with diminished neovascularization in an ischemic hindlimb model with less accumulation of CD34(+) progenitor cells in the ischemic muscle with or without local overexpression of SDF-1. Intravenous injection of BMCs from old mice homed less efficiently to ischemic muscle and stimulated significantly less neovascularization compared with the BMCs from young mice. Transplantation of old BMCs into young mice did not reconstitute CXCR4 functions suggesting that the defects were not reversible by changing the environment. We conclude that defects of basal and calcium-regulated functions of the CXCR4/SDF-1 axis in BMCs contribute significantly to the age-related loss of vasculogenic responses.

Enhanced cell therapy strategy to treat chronic limb-threatening ischemia.

Intermittent programmed compression of the chronically ischemic limb is associated with arteriogenesis. However, progenitor cell elements contributing to this neovascularization are typically diminished in number and function in the elderly dysvascular patient, particularly in the presence of diabetes, renal insufficiency, and cardiac disease. Granulocyte-colony stimulation factor (G-CSF) dramatically boosts the circulating progenitor cell count. G-CSF was administered in 2 patients being treated for ischemic wounds with an intermittent programmed pneumatic compression device (PPCD). Both had comorbidities associated with diminished circulating progenitor cell counts. Remarkable clinical, hemodynamic, and angiographic improvement was observed. Further study of this synergistic strategy is warranted.

Statin and stromal cell-derived factor-1 additively promote angiogenesis by enhancement of progenitor cells incorporation into new vessels.

Angiogenesis requires the mobilization of progenitor cells from the bone marrow and homing of progenitor cells to ischemic tissue. Statins facilitate the former, and the chemokine stromal cell-derived factor-1 (SDF-1) enhances the latter. Their combined influence on angiogenesis was studied in vivo in the ischemic hindlimb C57BL/6 mouse model. The ischemic to non-ischemic perfusion ratio increased from 0.29 +/- 0.02 immediately after femoral excision to 0.51 +/- 0.10 three weeks after the surgery in the mice treated with either fluvastatin or SDF-1 alone, which is significantly better than the control (0.38 +/- 0.05, p < .05, n = 6). The combined use of fluvastatin and SDF-1 further improved the reperfusion ratio (0.62 +/- 0.08, p < .05). More cell proliferation, less apoptosis, enhanced bone marrow-derived endothelial progenitor cell (EPC) incorporation and higher capillary density were observed in ischemic tissue treated with both statin and SDF-1. In vitro mono-treatment with either fluvastatin (100 nM) or SDF-1 (100 ng/ml) facilitated EPC proliferation and migration, inhibited EPC apoptosis, enhanced expression of matrix metalloproteinase-2 (MMP-2) and -9 (MMP-9), and increased Akt phosphorylation and nitric oxide production. These effects were significantly augmented by the two agents together and ablated by inhibitors of either Akt or nitric oxide synthase (NOS). In conclusion, statin and SDF-1 additively enhance progenitor cell migration and proliferation and down-regulate EPC apoptosis, resulting in improved reperfusion via activation of the Akt/NOS pathway and up-regulation of MMP-2 and MMP-9 expression.

Blood-derived smooth muscle cells as a target for gene delivery.

OBJECTIVE: To examine the feasibility of using blood-derived smooth muscle cells (BD-SMCs) as a target for to deliver therapeutic proteins. MATERIALS AND METHODS: Mononuclear cells (MNC) were isolated from peripheral blood. The outgrowth colonies from MNC culture were differentiated into BD-SMCs in media containing platelet-derived growth factor BB. Phenotypic characterization of BD-SMCs was assessed by immunocytochemistry. Cell proliferation, gene transfer efficiency with a retroviral vector, apoptosis, and the biological activity of the transduced gene product from the BD-SMCs were evaluated in vitro and in vivo in comparison with vascular derived SMC (VSMCs). RESULTS: BD-SMCs stained positive for SMC markers. No significant difference was observed between BD-SMCs and VSMCs in cell proliferation, migration, adhesiveness, and gene transfer efficiency. After BD-SMCs were transduced with a retroviral vector carrying the secreted alkaline phosphatase gene (SEAP), 174 +/- 50 mug biologically active SEAP was produced per 10(6) cells over 24 hours. After injecting 5 x 10(6) cells expressing SEAP intravenously into rabbits, SEAP concentration increased significantly in the circulation from 0.14 +/- 0.04 mug/ml to 2.34 +/- 0.16 mug/ml 3 days after cell injection (P < .01, n = 3). Circulating levels of SEAP decreased to 1.76 mug /ml 1 week later and remained at this level up to 8 weeks, then declined to pre-cell injection level at 12 weeks. VSMC in vivo gene expression data were equivalent. CONCLUSION: BD-SMCs have similar characteristics to mature VSMCs and can be used as a novel target for gene transfer to deliver a therapeutic protein.

Stromal cell-derived factor-1 enhances pro-angiogenic effect of granulocyte-colony stimulating factor.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) mobilizes bone marrow mononuclear cells into the peripheral circulation. Stromal cell-derived factor-1 (SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue. We hypothesize that SDF-1 will boost the pro-angiogenic effect of G-CSF. METHODS AND RESULTS: NIH 3T3 cells retrovirally transduced with SDF-1alpha gene (NIH 3T3/SDF-1) were used to deliver SDF-1 in vitro and in vivo. Endothelial progenitor cells (EPCs) co-cultured with NIH 3T3/SDF-1 cells using cell culture inserts migrated faster and were less apoptotic compared to those not exposed to SDF-1. NIH 3T3/SDF-1 (10(6) cells) were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. G-CSF (25 mug/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system. The perfusion ratio of ischemic/non-ischemic limb increased to 0.57+/-0.03 and 0.50+/-0.06 with the treatment of either SDF-1 or G-CSF only, respectively, 3 weeks after surgery, which was significantly higher than the saline-injected control group (0.41+/-0.01, P<0.05). Combined treatment with both SDF-1 and G-CSF resulted in an even better perfusion ratio of 0.69+/-0.08 (P<0.05 versus the single treatment groups). Mice were sacrificed 21 days after surgery. Immunostaining and Western blot assay of the tissue lysates showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1. CD34(+) cells were detected with immunostaining, capillary density was assessed with alkaline phosphatase staining, and the apoptosis of muscle cells was viewed using an in situ cell death detection kit. More CD34(+) cells, increased capillary density, and less apoptotic muscle cells were found in both G-CSF and SDF-1 treated group (P<0.05 versus other groups). CONCLUSION: Combination of G-CSF-mediated progenitor cell mobilization and SDF-1-mediated homing of EPCs promotes neovascularization in the ischemic limb and increases the recovery of blood perfusion.

Endovascular repair of thoracic aortic tears.

BACKGROUND: Standard treatment of traumatic thoracic aortic transection (TTAT) is open repair by left thoracotomy with or without the use of partial cardiopulmonary bypass. However, open repair is associated with high rates of morbidity and mortality, particularly in multiply injured trauma patients. We reviewed our experiences of endovascular repair of acute TTAT. METHODS: Between February 2001 and February 2006, 30 patients (male 24, female 6, mean age 43 years) who had sustained severe blunt trauma with multiple injuries (mean injury severity score = 42) underwent endovascular repair for TTAT. Devices used included commercially available proximal abdominal aortic extension cuffs and thoracic stent-grafts. Either low dose or no systemic heparin was used. Arterial access was obtained by femoral-iliac cutdown (n = 19) or completely percutaneous through the femoral artery (n = 11). Mean follow-up was 11.6 months (range, 1 to 48 months). RESULTS: Technically success was achieved in 100% of patients, as determined by angiographic and computed tomographic (CT) scan exclusion of TTAT. Mean operating time was 132 minutes. Mean blood loss was 300 cm3. Three patients had complications: 1 iliac artery rupture, 1 cerebellar stroke, and 1 partial stent collapse. There were 2 perioperative deaths. There were no instances of procedure-related paralysis. Clinical and CT follow-up did not reveal evidence of endoleak, stent migration, or late pseudoaneurysm formation. CONCLUSIONS: The adaptation of commercially available stent-graft devices to treat TTAT is technically feasible, and can be performed with low rates of morbidity and mortality. The long-term durability of endovascular repair of TTAT remains unknown, but early and midterm results appear promising.

Effect of tissue plasminogen activator on vascular smooth muscle cells.

OBJECTIVE: Engineered overexpression of tissue plasminogen activator (tPA) in vascular cells has been proposed as a means to decrease intravascular thrombosis; however, tPA gene transfer has augmented intimal hyperplasia in vivo in some studies. The purpose of this study was to define in vitro the effect of tPA gene transfer on smooth muscle cells (SMCs). METHODS: Human SMCs were retrovirally transduced with the tPA gene (SMCs/tPA). RESULTS: In the absence of plasminogen, no statistical differences in proliferation, migration, and morphology were observed between SMCs/tPA and SMCs. In the presence of plasminogen, many differences became apparent. Matrix metalloproteinase-2 (MMP-2) activation was 10-fold higher in SMCs/tPA than in SMCs. This activation was inhibited by aprotinin, a plasmin inhibitor. Collagen degradation increased sevenfold in SMCs/tPA. SMCs/tPA contracted dramatically in the presence of plasminogen. This cell contraction, indicative of extracellular matrix degradation, was blocked by aprotinin and partially inhibited by MMP inhibitors. SMC/tPA-conditioned medium induced significantly more SMC proliferation. The migration of SMCs/tPA through a porous membrane significantly exceeded untransduced SMCs. CONCLUSIONS: Over-expression of tPA in SMCs results in increased extracellular matrix degradation and can promote cell proliferation and migration. This effect is mediated via plasmin, which further activates MMP-2. CLINICAL RELEVANCE: TPA has been clinically used as a thrombolytic agent in the treatment of acute thrombotic disorders. Transferring the tPA gene into vascular cells as a strategy of gene therapy has been proposed to enhance fibrinolytic capability and therefore inhibit thrombosis and restenosis after vascular interventions. The mechanism(s) by which tPA affects SMC proliferation and vascular remodeling has not been thoroughly characterized. This study unveils the relationship between thrombolytic activity and intimal hyperplasia by showing how the elevated expression of tPA affects the vascular remodeling. This study underscores that the overexpression of an enzyme thought beneficial to blood flow can potentially compromise blood flow by altering the biology of the cell engineered to express it. The results are important to the rational engineering of bioactive grafts with better patency. A new strategy to enhance the thrombolytic ability of a vascular surface without inducing excessive neointimal hyperplasia is proposed.

Endograft technology: a delivery vehicle for intravascular gene therapy.

PURPOSE: The purpose of this study was to determine whether vascular smooth muscle cells (SMCs) suffused into a bilayered stent graft retain and express a retrovirally transduced gene for 7 months in vivo. METHODS: SMCs harvested from dog jugular vein were retrovirally transduced to introduce genes for tissue plasminogen activator (t-PA) and beta-galactosidase. These cells were then suffused into a novel dual-layered Dacron graft and cultured for 36 to 48 hours. The grafts were mounted on a Palmaz stent and balloon- expanded in the infrarenal aorta of the SMC donor dogs (n = 6). Grafts were recovered at 1, 2, 3, 4, 5, and 7 months. A control endograft suffused with SMCs transduced with only the beta-galactosidase gene was placed in the dogs with grafts recovered at 2, 3, and 4 months. t-PA antigen concentration and expression were analyzed with an enzyme-linked immunosorbent assay. RESULTS: Retained engineered SMCs (blue nuclei) were identified in the explanted grafts, neointima, and underlying aorta with X-gal staining. The t-PA antigen concentration and t-PA activity from the SMCs recovered from the grafts remained elevated for the duration of the experiment (7 months) at levels significantly higher (3.7 +/- 0.2 ng/mL per 10(5) cells per 24 hours and 1.4 +/- 0.1 IU/mL per 10(5) cells per 24 hours) than in control endografts (0.5 +/- 0.03 ng/mL per 10(5) cells per 24 hours and 0.07 +/- 0.00 IU/mL per 10(5) cells per 24 hours; P <.001). No graft stenosis was observed. CONCLUSION: Retrovirally engineered vascular SMCs survived the implantation trauma, repopulated each graft, migrated into the underlying aorta, and expressed the transduced genes for the 7-month duration of the experiment. This bilayered Dacron endograft model provides a platform to study direct intravascular gene therapy.

Smooth muscle cells improve endothelial cell retention on polytetrafluoroethylene grafts in vivo.

OBJECTIVE: We investigated the influence of smooth muscle cells (SMC) on endothelial cell (EC) retention on polytetrafluoroethylene (PTFE) grafts and the effect of SMC seeding on intimal hyperplasia in vivo in a rabbit model. METHODS: Fibronectin-coated PTFE grafts (4 mm diameter) were seeded with either EC alone, SMC alone, or SMC followed 24 hours later by EC. The grafts were connected to an extracorporal aortic shunt for 1 hour or were individually implanted for 1, 30, and 100 days into the infrarenal aorta as an end-to-side bypass graft. The number of retained cells was compared at 1 hour and at 1 day after implantation. Neointimal thickness was measured 30 and 100 days after implantation. RESULTS: After 1-hour exposure to blood flow, EC retention rate was greater (P <.005) if seeded on top of SMC (98% +/- 2%; n = 8) versus being seeded alone (65 +/- 11%; n = 8). SMC retention rate was 95 +/- 5% (n = 8) when seeded alone. Similar cell retention was obtained 1 day after implantation. After 30-day implantation the neointima was thicker in grafts seeded with EC and SMC (282 +/- 136 microm; n = 3) than with EC only (52 +/- 45 microm; n = 3; P <.001). However, the neointimal thickness for dual-cell-seeded grafts (126 +/- 60 microm; n = 3) was not significantly different (P =.09) from EC-seeded grafts (79 +/- 48 microm; n = 3) after 100-day implantation. CONCLUSION: EC retention on PTFE grafts in vivo is improved if seeded over a layer of SMC. Further studies are needed to determine whether overlying EC modulate proliferation of underlying SMC.

Late clinical presentation of femoral artery occlusion after deployment of the angio-seal closure device.

We report a case of late femoral artery thrombosis after deployment of the Angio-Seal closure device. The unusual late clinical presentation, aggressive anticoagulation which likely delayed clinical symptoms and the challenging surgical findings are discussed. Physicians need to be alert for possible complications arising late after deployment of closure devices and vascular surgeons must be familiar with the design of these devices since they may be required to repair a variety of arterial injuries associated with their use.

Immediate repair compared with surveillance of small abdominal aortic aneurysms.

BACKGROUND: Whether elective surgical repair of small abdominal aortic aneurysms improves survival remains controversial. METHODS: We randomly assigned patients 50 to 79 years old with abdominal aortic aneurysms of 4.0 to 5.4 cm in diameter who did not have high surgical risk to undergo immediate open surgical repair of the aneurysm or to undergo surveillance by means of ultrasonography or computed tomography every six months with repair reserved for aneurysms that became symptomatic or enlarged to 5.5 cm. Follow-up ranged from 3.5 to 8.0 years (mean, 4.9). RESULTS: A total of 569 patients were randomly assigned to immediate repair and 567 to surveillance. By the end of the study, aneurysm repair had been performed in 92.6 percent of the patients in the immediate-repair group and 61.6 percent of those in the surveillance group. The rate of death from any cause, the primary outcome, was not significantly different in the two groups (relative risk in the immediate-repair group as compared with the surveillance group, 1.21; 95 percent confidence interval, 0.95 to 1.54). Trends in survival did not favor immediate repair in any of the prespecified subgroups defined by age or diameter of aneurysm at entry. These findings were obtained despite a low total operative mortality of 2.7 percent in the immediate-repair group. There was also no reduction in the rate of death related to abdominal aortic aneurysm in the immediate-repair group (3.0 percent) as compared with the surveillance group (2.6 percent). Eleven patients in the surveillance group had rupture of abdominal aortic aneurysms (0.6 percent per year), resulting in seven deaths. The rate of hospitalization related to abdominal aortic aneurysm was 39 percent lower in the surveillance group. CONCLUSIONS: Survival is not improved by elective repair of abdominal aortic aneurysms smaller than 5.5 cm, even when operative mortality is low.

Injury induced neointima formation and its inhibition by retrovirus-mediated transfer of nitride oxide synthase gene in an in-vitro human saphenous vein culture model.

Human saphenous veins were cultured to characterize neointima formation and feasibility of gene transfer to inhibit the intimal proliferative response to injury. Mechanical injury was introduced by abrading the luminal surface of the vein patch with a sterile cotton bud. Both injured and non-injured vein patches were cultured and transduced with retroviral vectors carrying marker or therapeutic genes. After a 14-day culture, the thickness of the intimal layer of non-injured vein patches reached 90+/-28 microm at the edge and 61+/-22 microm at the center (n=29) from the original 22+/-12 microm at harvest (n=6, P=0.02). Mechanical injury to the intimal surface prior to culture resulted in an exaggerated proliferative response. The intimal thickness of injured vein patches increased from 3.4+/-1 microm right after injury to 128+/-23 microm (n=12, P<0.001) at the edge after 14-day culture. Genes were transduced efficiently into a luminal layer of cultured veins using a pseudotyped murine leukemia viral vector. Transduction of gene encoding nitric oxide synthase resulted in reduction of neointima formation to 33+/-7 microm (n=12) at the edge after 14-day culture compared to 90 microm (P<0.01) seen in untransduced non-injured vein patches. Marker gene transduction did not alter intimal proliferative response or its immunohistochemical profile. The data suggest that cultured vein can be used as a model for studying the effects of injury to blood vessels and to evaluate the effects of candidate therapeutic genes.