Functional arteries grown in vitro.

A tissue engineering approach was developed to produce arbitrary lengths of vascular graft material from smooth muscle and endothelial cells that were derived from a biopsy of vascular tissue. Bovine vessels cultured under pulsatile conditions had rupture strengths greater than 2000 millimeters of mercury, suture retention strengths of up to 90 grams, and collagen contents of up to 50 percent. Cultured vessels also showed contractile responses to pharmacological agents and contained smooth muscle cells that displayed markers of differentiation such as calponin and myosin heavy chains. Tissue-engineered arteries were implanted in miniature swine, with patency documented up to 24 days by digital angiography.

Antiserum to lymphocytes: prolonged survival of canine renal allografts.

A horse immunized with dog lynmphocytes produced an antiserum which agglutinated canine lymphocytes in vitro and caused prolonged lymphopenia in dogs in vivo. Renal transplants in dogs treated with this antiserum survived for long periods, two of the grafts surviving beyond 350 days with normal function and histologic appearance.

Sequencing of cDNA from 50 unrelated patients reveals that mutations in the triple-helical domain of type III procollagen are an infrequent cause of aortic aneurysms.

Detailed DNA sequencing of the triple-helical domain of type III procollagen was carried out on cDNA prepared from 54 patients with aortic aneurysms. The 43 male and 11 female patients originated from 50 different families and five different nationalities. 43 patients had at least one additional blood relative who had aneurysms. Five overlapping asymmetric PCR products, covering all the coding sequences of the triple-helical domain of type III procollagen, were sequenced with 28 specific sequencing primers. Analysis of the sequencing gels revealed only two nucleotide changes that altered the structure of the protein. One was a substitution of threonine for proline at amino acid position 501 and its functional importance was not clearly established. The other was a substitution of arginine for an obligatory glycine at amino acid position 136. In 40 of the 54 patients, detection of a polymorphism in the mRNA established that both alleles were expressed. The results indicate that mutations in type III procollagen are the cause of only about 2% of aortic aneurysms.

Fibulin-2 exhibits high degree of variability, but no structural changes concordant with abdominal aortic aneurysms.

We used conformation sensitive gel electrophoresis and direct sequencing of PCR products to screen for mutations in the cDNA for fibulin-2, an extracellular matrix protein, from 11 patients with abdominal aortic aneurysms and two controls. When compared with the published reference sequence, a total of 14 single-base sequence variations were detected. Seven of the changes were neutral in that they did not result in an amino acid substitution. There were five missense changes at sites not conserved between human and mouse, and two missense changes at sites conserved between human and mouse. All but two of the sequence variants studied were also present in an additional set of 102 control alleles analyzed. One of these two changes was a missense mutation, but it did not segregate with abdominal aortic aneurysms in the family, whilst the other change was neutral. In conclusion, fibulin-2 has a large number of sequence variations in comparison with our previous analyses of type III collagen, and these variations will be useful in association studies. There was an excellent overall agreement between direct sequencing of PCR-products and conformation sensitive gel electrophoresis.

Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins.

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.

The effectiveness and mechanism of collagen-induced topical hemostasis.

Microcrystalline collagen is a recently developed material possessing remarkable topical hemostatic properties. The potential utility of this substance for cardiovascular surgery was evaluated in a canine arterial bleeding model. A hemostatic protocol allowed comparison of initial and final hemostasis rates as well as the time required to accomplish hemostasis. The results of these studies revealed that microcrystalline collagen, or MCC, achieved hemostasis more frequently and rapidly than either pressure alone or oxidized cellulose cloth. MCC also was highly effective initially after large doses of heparin and in the presence of platelets with function rendered deficient by acetyl salicylate. Only in the presence of profound thrombocytopenia was any loss of initial effectiveness observed. Some delayed hemorrhage occurred in the heparin-treated groups leading to a lessened incidence of final hemostasis, a result intimately related to MCC's probable mechanism of action. However, even under the extreme conditions imposed by this severe experimental model, MCC remained effective in greater than 70 percent of the trials. On this basis MCC seems to possess great potential as a useful adjunct during cardiovascular surgical procedures.

Glutaraldehyde release from vascular prostheses of biologic origin.

The inhibition of growth by adult human venous endothelial cells in culture forms the basis of a sensitive assay of glutaraldehyde (GA) concentration, valid between 0 and 5 parts per million. This cytotoxicity assay was used to measure residual (unbound) GA in commercial vascular prostheses of biologic origin following manufacturer-recommended rinsing procedures, from which as much as 13.8 ppm GA per gram of tissue per 24 hours continued to be released after 1 month. A brief (1 hour) exposure of cultured endothelial cells to 2 ppm GA delayed growth, while continuous exposure to 4 ppm totally prevented growth for at least 12 days. Endothelial cells exposed to GA demonstrated a reduced efficiency of attachment to standard test surfaces, although prior GA treatment of these surfaces was not detrimental to subsequent cell attachment. GA release from vascular prostheses may contribute to their lack of endothelial cell coverage in human implants and may be indicative of collagen cross-link instability. If so, in vitro cytotoxicity may be helpful in identifying the potential for aneurysm formation in preserved biologic grafts.

Migration of adult human vascular endothelial cells: effect of extracellular matrix proteins.

An ideal vascular prosthetic graft should support rapid endothelial coverage and allow maximal rates of endothelial cell migration on its surface. To study this we modified and developed a method to measure rates of migration of adult human vascular endothelial cells (AHVECs) on test surfaces using an O-ring fence technique. We measured the rates of AHVEC migration on a variety of extracellular matrix proteins of interest because of their ability to support cell attachment. AHVECs, initially constrained in rings with an area of 0.62 cm2 (day 0), were allowed to migrate radially, and area coverage was measured after 7 days. The greatest areas of migration were seen on fibronectin (1.20 cm2) and gelatin (1.29 cm2) when compared with control (polystyrene [0.97 cm2]), p less than 0.01, p less than 0.05. Migration on laminin and prepared extracellular matrix from AHVEC was not statistically different from that of controls. Of extracellular matrix proteins studied in our system, a gelatin substrate in the presence of serum resulted in maximal rates of AHVEC migration.

Use of tritiated thymidine as a marker to compare the effects of matrix proteins on adult human vascular endothelial cell attachment: implications for seeding of vascular prostheses.

We have developed a technique to measure attachment of adult human vascular endothelial cells to test surfaces with tritiated thymidine used as a marker. With this technique, we measured attachment of adult human vascular endothelial cells to a series of extracellular matrix proteins, including fibronectin-coated (10 micrograms/cm2), laminin-coated (10 micrograms/cm2), and collagen-coated (1% gelatin) surfaces because of the role of these proteins in promoting cell attachment and growth. For a typical experiment, in the presence of serum, initial attachment (at 1 hour) was greatest on fibronectin-coated (63%) and gelatin-coated (60%) tissue culture plastic (polystyrene) and was least on laminin-coated (28%) or untreated polystyrene (18%). The data suggest that fibronectin, either alone, or with a more complex combination of extracellular components may need to be present on prosthetic surfaces to produce maximal cell attachment and subsequent growth to confluence in vivo. The described method of measuring attachment is independent of surface properties, ensures complete recovery of cells, and will allow systematic exploration of those properties that best support human endothelial cell attachment to vascular prosthetic surfaces.

Heparin fails to suppress intimal proliferation in experimental vein grafts.

Heparin has been shown to suppress both vascular smooth muscle cell proliferation in vitro and intimal hyperplasia in animal models of arterial injury. We investigated the effect of heparin on early postoperative smooth muscle cell proliferation and intimal thickening in vein grafts placed in the rat infrarenal aorta. Experimental animals (n = 6) received subcutaneous heparin (800 units/kg with levels monitored by activated factor X assay) every 12 hours for 3 days after surgery to coincide with the known period of maximal vascular wall DNA synthesis after injury. Control animals (n = 7) received identical vein grafts but no heparin. Grafts were harvested with perfusion fixation 14 days after insertion. Tritiated thymidine autoradiography was used to derive a mitotic index in regions of interest along the grafts, and computerized planimetric measurements of intimal and medial thickness were made in the same regions. Evans blue lumenal staining at harvesting revealed confluent endothelial coverage of both experimental and control grafts. The mitotic index of the vein graft's midsection and perianastomotic regions was significantly higher (p less than 0.005) than that of the native aorta in all animals, indicating cellular proliferation within all grafts, with no differences noted between heparinized and control animals. Similarly, both groups exhibited vein graft intimal and medial thickening at the anastomoses relative to the midsection of the vein graft (p less than 0.001). Heparin, administered in the equivalent of pharmacologic clinical doses, failed to suppress cellular proliferation and intimal hyperplasia in this model of vein grafting.

The effect of halothane anesthesia on platelet aggregation in vivo: decreased deposition on polytetrafluoroethylene arterial grafts in dogs.

The technique of gamma-imaging can be used to study the deposition of 111In-labeled platelets onto synthetic arterial grafts in vivo. Recent results suggested that platelet uptake on polytetrafluoroethylene (PTFE) grafts might depend on the choice of anesthetic. To evaluate the effect of anesthesia a series of experiments was performed in seven dogs wherein each animal served as its own control. The first of paired femoral or carotid PTFE grafts was inserted with the animal under pentobarbital anesthesia, and the graft was imaged for 90 minutes. A second graft was then inserted after at least 1 hour of halothane anesthesia. The mean activity ratio (describing platelet deposition) in the grafts inserted when only pentobarbital anesthesia was used was 7.04 +/- 0.55, compared to 1.20 +/- 0.07 in the grafts inserted with halothane anesthesia (P less than 0.01). Halothane anesthesia led to significantly decreased platelet uptake on canine PTFE arterial grafts. This effect was reversible, though not immediately, with no significant difference in graft activity noted by the day following surgery.

The value of electrocardiogram monitoring during treadmill testing for peripheral vascular disease.

In evaluating the arterial hemodynamics of the lower extremities in patients with peripheral vascular disease, the treadmill exercise test offers useful information concerning the peripheral circulation. The addition of electrocardiographic (EKG) monitoring during the treadmill examination provides a method of estimating the effectiveness of coronary circulation. In three groups of patients studied, the results demonstrated that EKG monitoring during the treadmill exercise test will reveal previously unsuspected arrhythmias and ischemia. Thus the safety of the patient is enhanced. In patients followed through the operative period, there was a high correlation between exercise-induced EKG changes and perioperative cardiac problems. The value of EKG monitoring during treadmill testing is clear and offers predictive information to the surgeon contemplating a revascularization procedure.

Intestinal ischemia complicating abdominal aortic surgery.

A 9-year experience with 2137 patients undergoing infrarenal abdominal aortic reconstruction was reviewed to determine both the incidence of intestinal ischemia and the clinical, anatomic, and technical factors associated with this complication of aortic surgery. A total of 24 (1.1%) patients had overt intestinal ischemia, documented by reoperation or endoscopic findings. Of these, colon ischemia occurred in 19 (0.9%) and small bowel ischemia developed in 5 (0.2%) patients. The incidence after elective operation for aneurysmal or occlusive disease did not differ, but patients with ruptured aneurysms and those undergoing reoperative procedures for total graft replacement were at higher risk. Preoperative angiography was most helpful in ascertaining risk. Ligation of a patent inferior mesenteric artery was the most common (74%) feature in patients with colon ischemia. With preexisting inferior mesenteric artery occlusion, impairment of collateral circulation was attributable to superior mesenteric artery disease, dissection or retractor injury, prior colon resection, or exclusion of hypogastric perfusion. Bloody diarrhea was the most frequent postoperative symptom and colonoscopy the most reliable means of diagnosis. One half of patients with colon ischemia required resection after late recognition of perforation. All cases of small bowel ischemia were related to superior mesenteric artery disease or injury or use of suprarenal clamping. The overall mortality rate was 25% but rose to 50% if bowel resection was required. Intestinal ischemia remains an infrequent but serious complication of aortic surgery. Despite a multifactorial cause, identification of patients at increased risk can lead to operative strategies to reduce its occurrence.

Acute arterial thrombosis of the lower extremity. Its natural history contrasted with arterial embolism.

We compared a group of 52 patients with acute lower extremity ischemia secondary to arterial thrombosis with a series of 220 patients with peripheral embolism who were seen at the Massachusetts General Hospital, Boston, from 1967 through 1980. The conditions of patients with arterial thrombosis were misdiagnosed as acute embolism at a rate of 20%. Mortality in the patients with embolism was significantly higher. While patients with acute embolism were routinely treated with embolectomy, two thirds of the patients with thrombotic occlusion required surgery during their initial hospitalization for ongoing severe ischemia. More than half of these operations were carried out as emergency procedures. Limb salvage in surgically treated patients with arterial thrombosis was inferior to that seen with embolism. Early revascularization for acute thrombosis was carried out with a 30-day patency rate of 82%. While the mortality associated with acute thrombosis was significantly lower than that seen with peripheral embolism, the risk of major amputation was 35%. There should be no reluctance to proceed with indicated vascular reconstruction in the setting of acute limb ischemia secondary to arterial thrombosis.

Postsurgical changes in arterial compliance.

Mismatch in elasticity between artery and graft, a possible contributing factor to graft failure, has traditionally been believed to be caused by the graft. Compliance of the artery and/or graft may change after surgery, resulting in a different compliance mismatch. To study the arterial contribution to compliance mismatch, we measured compliance-pressure (CP) curves noninvasively in the femoral arteries of five dogs. One femoral artery was then simply exposed, and the incision was closed. Repeated CP curves were obtained after 24 hours, and after 1, 2, 3, and 4 weeks. Compliance remained normal after 24 hours, but by one week compliance of the dissected arteries had dropped significantly at all pressures between 70 and 120 mm Hg. Compliance reached a minimum after two weeks and remained low until after four weeks. Postsurgical arterial stiffening causes a significant decrease of arterial-graft compliance mismatch and may suggest an upper limit for synthetic graft compliance.

Adult human vascular endothelial cell attachment and migration on novel bioabsorbable polymers.

We studied the ability of polymers from which vascular prostheses (VPs) are or could be fabricated to support attachment and migration of adult human vascular endothelial cells (AHVECs) in an in vitro system. Polymers included Mylar, Teflon, two novel bioabsorbable polymers with different biologic half-lives (BR1 and BR2), and an elastic nonabsorbable biocompatible polymer (EBN). Both BR1, BR2, and EBN supported AHVEC attachment well (56%, 62%, and 71%, respectively, of plated cells at one hour, respectively) but differed in their ability to support AHVEC migration. The AHVECs attached less well to Mylar and Teflon (39% and 21%), and in no case were AHVECs observed to grow or migrate on either of these polymers. This finding correlates with the clinical observation that VPs fabricated from Mylar and Teflon uniformly fail to develop an endothelial cell lining after implantation in vivo. Future VPs, fabricated from materials chosen for their ability to support AHVEC attachment, growth, and migration in vitro, might better support a spontaneous endothelial cell lining after implantation in humans.

Matched elastic properties and successful arterial grafting.

Clinical results from medium- and small-caliber arterial bypass grafts are unsatisfactory. Since elastic properties of grafts tested experimentally have been correlated with patency results, the compliance of the human femoral artery was compared with grafts currently in use: human saphenous vein (HSV), knitted Dacron (DAC), glutaraldehyde-treated umbilical cord vein (DBM), bovine heterograft, and expanded polytetrafluoroethylene (PTFE). This was correlated with clinical patency data for the different conduits in the femoropopliteal position. Increased patency correlated with a decreasing disparity between host artery and graft compliance. After two years, patency rates of the more compliant materials (HSV, DBM) exceeded 80%, while less than 45% of the incompliant grafts (DAC, PTFE) remained patent. Thus, clinical performance with synthetic grafts might be improved by use of prostheses in which the viscoelastic characteristics match those of arteries more closely.

Neurologic sequelae with internal carotid artery occlusion.

A retrospective review of the clinical course surrounding internal carotid artery occlusion (ICO) was undertaken in 97 patients with 106 instances of ICO. No neurologic symptoms could be attributed to more than half of the ICOs. Of the 52 symptomatic occlusions, 19 (37%) were associated with transient ischemic attacks and 33 (63%) with fixed strokes. Only 10% of all patients had permanent disabling neurologic sequelae. There was no correlation between development of neurologic symptoms and the side of the ICO, the presence or severity of contralateral carotid artery disease, or other risk factors. Women, however, were twice as likely as men to develop a fixed stroke with ICO. Seventy-six of these patients underwent reconstructive carotid surgery. Although there was a high rate of abnormal intraoperative findings with electroencephalographic monitoring (32 of 62 cases), with the use of intraoperative shunts there was no increase in the postoperative stroke complication rate (1.8%). These data suggest that the concern that ICO leads to serious fixed neurologic deficits may be overestimated, and that its presence does not adversely affect carefully performed contralateral carotid endarterectomy.

Femoropopliteal and tibioperoneal artery reconstruction using human umbilical vein.

We used 99 human umbilical vein (HUV) grafts for femoropopliteal or tibioperoneal artery reconstruction in 94 patients. Results were calculated according to the life-table method for several categories possibly influencing graft patency. For above-knee v below-knee distal anastomosis, there was no difference in patency or limb-salvage rates at 30 months. Comparison of good and poor runoff situations disclosed similar 30-month graft patency rates, but significantly better limb-salvage rates among patients with good runoff. Primary revascularization fared significantly better than secondary procedures in terms of patency and limb salvage. We found no significant differences between HUV and polytef (polytetrafluoroethylene) for above-knee anastomoses or good runoff situations. However, patency and limb salvage were better for HUV use in below-knee anastomosis and poor runoff situations. The HUV may offer advantages in long-term patency and limb salvage in selected patients.

Vascular complications of intra-aortic balloon counterpulsation.

Although the intra-aortic balloon pump (IABP) saves many lives, complication rates as high as 36% have been reported. Many of the complications require surgical attention. In 794 patients of the Massachusetts General Hospital in Boston, an IABP was inserted into either the femoral or iliac artery between Nov 1, 1968, and June 30, 1981. Eighty-seven major vascular complications occurred in 70 (8.8%) of them. Of those 70 patients, 95% denied previous symptoms of vascular disease, and 78% had had normal pulses before insertion. The two most common complications were limb ischemia (n = 36) and arterial trauma (n = 20). Other complications included sepsis (n = 14), wound problems (n = 10), and hemorrhage (n = 7). No limbs were lost. Analysis of each group of complications allowed specific conclusions to be drawn concerning prevention, early diagnosis, and the most appropriate treatment.