Sanford I. Roth

Transforming Growth Factor-β1 Fails to Stimulate Wound Healing and Impairs Its Signal Transduction in an Aged Ischemic Ulcer Model : Importance of Oxygen and Age

Clinical trials of exogenous growth factors in treating chronic wounds have been less successful than expected. One possible explanation is that most studies used animal models of acute wounds in young animals, whereas most chronic wounds occur in elderly patients with tissue ischemia. We described an animal model of age- and ischemia-impaired wound healing and analyzed the wound-healing response as well as the transforming growth factor (TGF)-beta1 effect in this model. Rabbits of increasing ages were made ischemic in the ear where dermal ulcers were created. Histological analysis showed that epithelium ingrowth and granulation tissue deposition were significantly impaired with increased age under ischemia. TGF-beta1 stimulated wound repair under both ischemic and non-ischemic conditions in young animals, although it showed no statistical difference in aged animals. Procollagen mRNA expression decreased under ischemic conditions and with aging. Neither TGF-beta1 nor procollagen alpha1(I) mRNA expression increased in response to TGF-beta1 treatment under ischemia in aged animals. Therefore, the wound-healing process is impaired additively by aging and ischemia. The lack of a wound-healing response to TGF-beta1 in aged ischemic wounds may play a role in the chronic wounds.

Accurate Three-Dimensional Reconstruction of Intravascular Ultrasound Data Spatially Correct Three-Dimensional Reconstructions

BACKGROUND The geometrical accuracy of conventional three-dimensional (3D) reconstruction methods for intravascular ultrasound (IVUS) data (coronary and peripheral) is hampered by the inability to register spatial image orientation and by respiratory and cardiac motion. The objective of this work was the development of improved IVUS reconstruction techniques. METHODS AND RESULTS We developed a 3D position registration method that identifies the spatial coordinates of an in situ IVUS catheter by use of simultaneous ECG-gated biplane digital cinefluoroscopy. To minimize distortion, coordinates underwent pincushion correction and were referenced to a standardized calibration cube. Gated IVUS data were acquired digitally, and the spatial locations of the imaging planes were then transformed relative to their respective 3D coordinates, rendered in binary voxel format, resliced, and displayed on an image-processing workstation for off-line analysis. The method was tested by use of phantoms (straight tube, 360 degrees circle, 240 degrees spiral) and an in vitro coronary artery model. In vivo feasibility was assessed in patients who underwent routine interventional coronary procedures accompanied by IVUS evaluation. Actual versus calculated point locations were within 1.0 +/- 0.3 mm of each other (n = 39). Calculated phantom volumes were within 4% of actual volumes. Phantom 3D reconstruction appropriately demonstrated complex morphology. Initial patient evaluation demonstrated method feasibility as well as errors if respiratory and ECG gating were not used. CONCLUSIONS These preliminary data support the use of this new method of 3D reconstruction of vascular structures with use of combined vascular ultrasound data and simultaneous ECG-gated biplane cinefluoroscopy.

Interaction between the insulin-like growth factor family and the integrin receptor family in tissue repair processes. Evidence in a rabbit ear dermal ulcer model.

We have determined previously that IGF-I is dependent on the presence of IGF binding protein-1 (IGFBP-1) to act as a wound healing agent. We sought to determine the mechanism whereby IGFBP-1 is able to enhance IGF-I bioactivity. As IGFBP-1 binds both the alpha5beta1 integrin as well as IGF-I in vitro, we asked which of the following interactions were important: (a) the ability of IGFBP-1 to interact with an integrin receptor, and/or (b) the binding of IGF-I by IGFBP-1. We used an IGF-1 analogue (des(1-3)IGF-I) with a > 100-fold reduction in affinity for IGFBP-1 as well as an IGFBP-1 mutant (WGD-IGFBP-1) which does not associate with the alpha5beta1 integrin to selectively abrogate each of these interactions. We also tested the ability of IGFBP-2, a related binding protein which has an arginine-glycine-aspartate sequence but does not associate with integrin family members, to enhance IGF-I bioactivity. Full-thickness dermal wounds were created on rabbit ears; various combinations of native IGF-I, native IGFBP-1, native IGFBP-2, and their respective analogues/mutants were applied to each wound. Wounds were harvested 7 d later for analysis. Only native IGF-I in combination with native IGFBP-1 was effective as a wound healing agent, enhancing reepithelialization and granulation tissue deposition by 64+/-5 and 83+/-12% over controls (P = 0.008 and 0.016, respectively). The same doses of IGF-I/WGD-IGFBP-1, des(1-3)IGF-I/IGFBP-1, and IGF-I/IGFBP-2 were ineffective. We propose that IGF-I physically interacts with IGFBP-1 and that IGFBP-1 also binds to an integrin receptor, most likely the alpha5beta1 integrin. This interaction is unique to IGFBP-1 as the closely related IGFBP-2 had no effect, a finding consistent with its inability to bind to integrin receptors. Our results suggest that activation of both the IGF-I receptor and the alpha5beta1 integrin is required for IGF-I to stimulate wound healing.

Analysis of Parathyroid Hormone and Its Fragments in Rat Tissues: CHEMICAL IDENTIFICATION AND MICROSCOPICAL LOCALIZATION

After intravenous injection of [(125)I]-iodo-parathyroid hormone in the rat, uptake of the hormone was greatest in the liver and kidneys. Uptake was rapid, reaching a maximal concentration by 4 and 8 min, respectively. Extracts, prepared from both these organs at intervals soon after the injection of intact hormone, showed three main radioactive peaks when samples were subjected to gel filtration under protein-denaturing conditions. The first peak coeluted with intact hormone. The second eluted at a position corresponding to the carboxy-terminal fragments previously described in plasma, and the last eluted at the salt volume of the column. Microsequence analysis of the radioiodinated fragments, a method that has proved valuable for chemically defining the circulating fragments resulting from metabolism of injected hormone, showed that extracts of liver and kidney, prepared at 4 and 8 min after injection of the intact hormone, contained different fragments. The radioiodinated fragments in liver extracts were identical to those previously reported in the plasma of rats and dogs, fragments resulting principally from proteolysis between positions 33 and 34, and 36 and 37 of the intact hormone. Although the same fragments were also present in the kidneys, they constituted less than 15% of the amount present in the liver. More than 50% of the labeled renal fragments consisted of a peptide whose amino-terminal amino acid was position 39 of the intact hormone, a fragment not present in plasma. The rate of appearance of radioiodinated fragments that were chemically identical to those in plasma was more rapid in the liver than in plasma. Correlation of these chemical analyses with studies of the localization of (125)I by autoradiography showed that at the times when the intact hormone and the carboxy-terminal fragments comprised nearly all of the (125)I-labeled moieties in the tissues, the proximal convoluted tubules of the kidney and sinusoidal lining cells of the liver, which probably are Kupffer cells, contained the highest concentration of (125)I. Preferential localization of immunoreactive parathyroid hormone to these tissue sites also was shown by immunoperoxidase staining in studies with unlabeled hormone. Our results suggest that, unless multiple renal mechanisms are present for release of hormonal fragments, one of which releases the circulating fragments preferentially, the liver, rather than the kidney, is principally responsible for generating the carboxy-terminal fragments in plasma after injection of intact hormone, and the Kupffer cells may contain the enzymes that hydrolyze parathyroid hormone.

Arterial imaging with a new forward-viewing intravascular ultrasound catheter, II. Three-dimensional reconstruction and display of data.

BackgroundCurrent intravascular ultrasound (IVUS) catheters provide transverse imaging at the level of the ultrasound transducer. This limits imaging to large-diameter segments without critical atherosclerotic narrowings. We have developed a prototype 20-MHz forward-viewing IVUS catheter that provides two-dimensional sector imaging distal to the catheter tip. A present limitation of this technique is that the catheter must be manually rotated to obtain multiple longitudinal views required to integrate the segment into a three-dimensional matrix. To overcome this, we have developed an algorithm that reconstructs these multiple two-dimensional forward-viewing IVUS images into a three-dimensional matrix for more complete depiction of the segment distal to the ultrasound catheter. This algorithm allows display and multidimensional slicing of the three-dimensional reconstruction. Methods and ResultsTo test our algorithms, five arterial segments (three canine aortas, two human femoral arteries) were evaluated in vitro. In each segment, 36 forward-viewing longitudinal slices were collected, digitized, processed, and reoriented to produce a three-dimensional reconstruction (3DR) matrix. The matrix data were sliced into parallel transverse sections and compared with morphometric interpretation of histological sections (Histo). As a result, image data could be reconstructed for a distance of 2.0 cm ahead of the catheter. 3DR easily demonstrated wall and luminal morphology and provided transverse IVUS images comparable to the histological specimens. A good correlation was noted between Histo- and 3DR-determined luminal diameters (LD) and luminal areas: 3DR LD=1.4 Histo LD−0.4, r = .86; 3DR LD=0.7±0.20 cm (mean±SD); and Histo LD=0.7±0.13 cm. ConclusionsThese preliminary data demonstrate the feasibility of 3DR of forward-viewing IVUS data. This method allows rapid, detailed analysis of diseased arterial segments previously unavailable with standard IVUS and may permit better targeting of interventional techniques.

Anatomy and histological characteristics of the spinoglenoid ligament

The spinoglenoid (inferior transverse scapular) ligament, when present, is located at the spinoglenoid notch. The ligament originates on the spine of the scapula and inserts on the superior margin of the glenoid neck. Because of discrepancies in the literature, we sought to determine its prevalence and to define its histological characteristics. We dissected 112 shoulders of seventy-six cadavera and classified the ligament as absent or an insubstantial structure, a thin fibrous band (type I), or a distinct ligament (type II). We found no distinct ligamentous structure in twenty-two shoulders (20 percent), a type-I ligament in sixty-eight shoulders (20 percent), a type-I ligament in sixty-eight shoulders (61 percent), and a type-II ligament in twenty-two shoulders (20 percent). Overall, ninety (80 percent) of the shoulders had a fibrous band of tissue that, together with the spine of the scapula, formed a narrow fibro-osseous tunnel through which the suprascapular nerve traveled. The bone-spinoglenoid ligament-bone complexes from three specimens were analyzed histologically. There were two type-I ligaments and one type-II ligament; all three ligaments were composed of collagen fibers. One type-I ligament and the type-II ligament demonstrated Sharpey fibers at their origin on the spine of the scapula. The other type-I ligament attached to the spine of the scapula through the periosteum. All three ligaments inserted into the periosteum of the glenoid neck. CLINICAL RELEVANCE: The spinoglenoid ligament may be clinically relevant in two respects. First, the ligament may limit mobilization and advancement of the infraspinatus tendon during repair of a massive tear of the rotator cuff, placing the distal part of the suprascapular nerve at risk. Second, the spinoglenoid ligament represents a potential site for nerve entrapment, particularly with the added stress of traction that can occur with overhead athletic activities.

Parathyromatosis in hyperparathyroidism

Recurrent hyperparathyroidism after parathyroidectomy may present a difficult diagnostic problem. A rare etiology is parathyromatosis (multiple nodules of hyperfunctioning parathyroid tissue scattered through the neck and mediastinum) due to spillage of otherwise benign parathyroid tissue during surgery. We present a case of recurrent hyperparathyroidism and parathyromatosis due to tissue spillage during surgical removal of probable double adenomas, a rare cause of primary hyperparathyroidism. Thus, parathyromatosis must be included in the differential diagnosis of recurrent or persistent hyperparathyroidism, distinguished from parathyroid carcinoma by histologic criteria. The surgeon must be careful of parathyroid spillage during surgery, even of benign tumors of the parathyroids.

Cryoglobulinemia and cutaneous leukocytoclastic vasculitis associated with hepatitis C virus infection

Hepatitis C virus infection is a frequent cause of non-A, non-B hepatitis worldwide. Resultant morbidity is significant; chronic liver disease develops in 50% of infected persons. Since serologic testing has become available there have been several reports of cutaneous findings in association with hepatitis C virus infection, including vasculitis, cryoglobulinemia, urticaria, and lichen planus. We describe a patient with cryoglobulinemia, chronic cutaneous leukocytoclastic vasculitis, and hepatitis C virus infection. Hepatitis C virus infection should be included in the differential diagnosis of the causes of cryoglobulinemia and leukocytoclastic vasculitis.

Effects of insulin-like growth factor-I and insulin-like growth factor binding protein-1 on wound healing in a dermal ulcer model†

The effects on wound healing of insulin‐like growth factor‐I with and without insulin‐like growth factor binding protein‐1 were studied in a rabbit ear dermal ulcer model under both nonischemic and ischemic conditions. Wounds 6 mm in diameter were made on the ventral surface of rabbit ears for a total of 272 wounds in the nonischemic group and 77 wounds in the ischemic group. Insulin‐like growth factor‐I in varying doses (1 to 43 µg) and in combination with varying molar ratios of the binding protein were added at time of wounding to each wound. Wounds were analyzed histomorphometrically on day 7 after wounding. We found that insulin‐like growth factor‐I or binding protein alone at varying doses did not have any effects on wound healing parameters. Low to moderate doses (1 µg and 4 µg, respectively) of the combination of insulin‐like growth factor‐I with the binding protein in a molar ratio of 5:1 or 11:1 showed a 52% increase (p < 0.05) in new granulation tissue in the nonischemic model compared with controls but did not significantly augment new granulation tissue formation in the ischemic wound model. A high dose (43 µg) at a 10:1 molar ratio of growth factor to binding protein was required to elicit significantly enhanced wound healing in ischemic wounds. These results indicate that insulin‐like growth factor binding protein‐1 modulates the effects of insulin‐like growth factor‐I in promoting wound healing in vivo and that the combination is a highly effective vulnerary compound with effects comparable in magnitude with other growth factors previously tested in this model.

The uptake of horseradish peroxidase by the conjunctival epithelium of the guinea-pig

It has been suggested that in immediate hypersensitivity of the guinea-pig conjunctiva, induced by the topical application of an antigen, the antigen is selectively taken up by the conjunctiva-associated lymphoid tissue. We tested this hypothesis by applying horseradish peroxidase (HRP) to the guinea-pig conjunctiva and studying its uptake by light- and electron microscopy. As early as 30 min after the application of the HRP, precipitate indicating the presence of HRP could be seen in the epithelial cells in membrane limited granules in the non-lymphoid and lymphoid epithelium. There was no selective uptake of HRP by the lymphoid-associated epithelial cells. We hypothesize that the epithelial cells of the conjunctiva, in contrast to those of the small intestine, phagocytose antigen and transfer it directly to the substantia propria of the conjunctiva for local immunologic processing.