Biomechanical properties of veins cultured in vitro under elevated internal pressure.

BACKGROUND: The venous response to elevated blood pressure (BP) is of major importance because it is closely related to the etiology of venous diseases and the competency of vein grafts. In vitro culture experiments may provide useful information on the function of vein grafts because it is easier to separate mechanical and hemodynamic effects from other systemic influences compared to in vivo experiments. OBJECTIVE: To study the effects of BP elevation on wall dimensions and mechanical properties of in vitro cultured veins. METHODS: Rabbit femoral veins were cultured in vitro under internal pressures of 1 to 50 mmHg for 1 week, and their wall dimensions, biomechanical properties, and histology were determined. RESULTS: No significant differences were observed in internal vein diameter and wall thickness among vessels cultured at 10-50 mmHg compared to non-cultured control vessels. For an internal pressure of 10 mmHg applied to vessels during culture (equivalent to in vivo working BP), wall circumferential stress was maintained within control levels. There were no significant effects of pressure on basal tone and contractility of vascular smooth muscle and vascular compliance. CONCLUSIONS: The in vitro results were essentially similar to those obtained from previous in vivo animal experiments, indicating that in vitro tissue culture techniques are applicable to studies of venous remodeling.

Remodeling of the arterial wall: Response to restoration of normal blood flow after flow reduction.

BACKGROUND: Although many studies have shown that arteries change diameter in response to chronic change in blood flow (BF), keeping wall shear stress (WSS) at physiologically normal levels, relatively little is known about the effects of flow restoration after flow reduction and also the role of vascular smooth muscle (VSM) during such a remodeling process. OBJECTIVE: To elucidate the biomechanical responses of the arterial wall to the restoration of normal BF after flow reduction and compare the results with our previous results observed in response to decreased BF alone. METHODS: Carotid artery BF in the Wistar rat was decreased by ligation and then restored to normal levels by release of the ligation. The effects of BF changes on the biomechanical properties of the carotid arterial wall were determined from measurements of diameters and pressures of excised artery segments. RESULTS: During BF reduction and restoration, WSS was maintained at physiological levels by changes in the internal diameter. No significant changes in the incremental elastic modulus were found in response to changes in BF. VSM tone was significantly enhanced during the changes in BF. CONCLUSIONS: Arteries change diameters in response to BF reduction and also flow restoration to normal after flow reduction, keeping WSS at physiologically normal levels. The lack of changes in vascular elasticity suggests that there were no significant changes in major wall constituents, such as elastin and collagen. VSM may play the dominant role in observed arterial remodeling and adaptation.

Remodeling of arterial wall: Response to changes in both blood flow and blood pressure.

Many studies have been performed on arterial responses to chronic changes in blood flow (BF) and blood pressure (BP). However, little is known about the effects of simultaneous changes in BF and BP. The present study was carried out to know biomechanical responses of arterial wall to the combination of increased BP, i.e. hypertension (HT), with lower or higher BF than normal, and the results were compared with those observed under normal BP, i.e. normotension (NT), combined with these BF conditions. Eight weeks old rats were subjected to BF and/or BP changes for 8 weeks until 16 weeks of age. Systemic HT was induced by the constriction of one of the renal arteries (Goldblatt HT), while BF in the CCA was reduced and increased by the constriction of the ipsilateral CCA and the ligation of the contralateral CCA, respectively. The internal diameter of the target CCA was significantly larger in higher BF groups than in lower BF ones irrespective of HT. Wall shear stress (WSS) was normalized by such compensatory changes in the diameter. Wall thickness was significantly larger in HT rats than in NT ones regardless of BF, and the wall hypertrophy contributed to restore wall hoop stress to normal level. Basal vascular tone, arterial stiffness, and wall elastic modulus were significantly larger in HT than in NT independently of BF changes. However, only in HT/lower BF group, WSS and vascular smooth muscle-activated vascular contraction were smaller than in the other groups, possibly because of wall hypertrophy induced by HT.

Effects of hypothermia on the mechanical behavior of rabbit femoral arteries.

The need to better understand the effects of non-physiological temperatures on arterial wall behavior is becoming more important because of the increased clinical use of hypothermal and hyperthermal treatments. The present study was performed to examine the effects of temperature on the mechanical behavior of femoral arteries excised from rabbits. Among 17, 27, 37, and 42°C, there were no significant differences in their diameter, stiffness, and P-D relations under the physiologically normal, control condition, although the arterial diameter was slightly smaller at 42°C than at the other three temperatures. Under the SMC-activated condition, on the other hand, we observed significant effects of temperature. For example, arterial diameter at 100mmHg was significantly larger at 17 and 27°C and smaller at 42°C compared with 37°C. Arterial stiffness at 40mmHg were significantly smaller at 17 and larger at 42°C than at 37°C, while the stiffness at 160mmHg were significantly larger at 17°C than at 37°C; however, there were no significant differences in the stiffness at 100mmHg among the four temperatures. Arterial contraction induced by SMC-activation was significantly different between 37°C and the other three temperatures; both of the maximum diameter response and diameter response at 100mmHg were significantly smaller at 17 and 27°C and larger at 42°C compared with 37°C. These results indicate that in the hypothermic range under the control condition, arteries are dilated when cooled, while they are constricted when heated. On the other hand, arterial response to SMC activation is significantly affected by the alterations of temperature. These results indicate that in the hypothermic range under the control condition, arteries are dilated when cooled, while they are constricted when heated. On the other hand, arterial response to the activation of vascular smooth muscle cells is significantly affected by the alteration of temperature. As the mechanical behavior of arterial wall is significantly influenced by temperature, this should be considered in the development of therapeutic methods and techniques for cardiovascular diseases.

Stiffness of Intact Endothelial Cells From Fresh Aortic Bifurcations of Atherosclerotic Rabbits-Atomic Force Microscopic Study.

Stiffness of intact endothelial cells (ECs) in the abdominal aorta (AA) and in the medial and lateral wall of the common iliac artery (CIA(Medial) and CIA(Lateral), respectively), which were freshly obtained from cholesterol-fed rabbits, were measured with an atomic force microscopic indentation method. In the areas away from atherosclerotic plaques (Off-plaque), ECs were significantly stiffer in CIA(Medial) than in the other two locations; this result was similar to that from normal diet-fed animals. On the other hand, there were no significant differences in the stiffness of ECs located on atherosclerotic plaques (On-plaque) among the three sites; the stiffness was equal to those in "Off-plaque" wall of CIA(Lateral) and AA. Moreover, the stiffness of ECs covering plaques decreased with the progression of atherosclerosis. The precise quantification of the stiffness of vascular ECs would provide a better understanding of cellular remodeling and adaptation in atherosclerosis. J. Cell. Physiol. 232: 7-13, 2017. © 2016 Wiley Periodicals, Inc.

Age-related changes of wall composition and collagen cross-linking in the rat carotid artery - In relation with arterial mechanics.

In association with age-related changes in arterial wall mechanics, the composition of connective tissues, the fraction and size of vascular smooth muscle cells (SMCs), and the degree of collagen cross-linking were studied with common carotid arteries harvested from 8, 16, 32, and 64 week-old Wistar rats. For histomorphometric studies, each arterial segment was fixed under in vivo operating force condition, and then sequentially sliced into thin specimens, followed by selective staining for the observation of collagen, elastin, and SMCs. Then, the fraction of each component, and the number and size of SMCs were determined with an image analyzer. The content of collagen and elastin, their ratio, and the number and the area fraction of SMCs showed no significant correlations with age, while the density and the size of SMCs were significantly smaller and larger, respectively, in 64 week-old animals than in the others. The results of collagen and elastin cannot explain the biomechanical data obtained in our previous study using the same animal model, which showed that the elastic modulus and wall stiffness were significantly larger in 64 week-old animals compared to younger ones. To investigate the reason for the discrepancy between the histological and the biomechanical results, a hydrothermal isometric tension method was applied to the analysis of the cross-linking of collagen, and we found that the amount of cross-links was significantly greater in 64 week-old arteries than in the others. This result corresponded well with the biomechanical results, and therefore the higher wall stiffness and elastic modulus in older arteries might be ascribed to their larger amount of collagen cross-links.

Composition of connective tissues and morphometry of vascular smooth muscle in arterial wall of DOCA-salt hypertensive rats - In relation with arterial remodeling.

Hypertension (HT) was induced in Wistar rats aged 16 and 48 weeks by a deoxycortico-sterone acetate (DOCA)-salt procedure. Common carotid arteries were resected 16 weeks after, and their histological specimens were selectively stained for observations of collagen, elastin, and vascular smooth muscle (VSM) cells. Then, the fractions of collagen and elastin and their radial distributions, and the size and number of VSM cells were determined with an image analyzer. These results were compared with the results from age-matched, non-treated, normotensive (NT) animals and also with those from our previous biomechanical studies. In both age groups, there were no significant differences in the fractions of collagen and elastin, and the ratio of collagen to elastin content between HT and NT arteries. These results correspond well with our previous biomechanical results, which showed no significant difference in wall elasticity between HT and NT vessels. Moreover, in the innermost layer out of 4 layers bordered with thick elastic lamellae, the fraction of collagen was significantly greater in HT arteries than in NT ones, which is attributable to HT-related stress concentration in the layer. VSM cells were significantly hypertrophied and their content was increased by HT, although their total number in the media remained unchanged. The increased size and content of cells correspond to the enhancement of vascular tone and contractility in HT arteries.

Correlation Between Recurrent Subconjunctival Hemorrhages and Conjunctivochalasis by Clinical Profile and Successful Surgical Outcome.

OBJECTIVE: To characterize the clinical profile of patients with recurrent subconjunctival hemorrhages (SCHs) and evaluate the effect of conjunctivochalasis (CCh) surgery on disease recurrences. METHODS: Three hundred and sixty-two patients with SCHs (mean age, 56.4±16.0 years) were enrolled in this multicenter epidemiologic study. The severity of CCh, lifestyle at the time of SCH onset, and the frequency of previous SCHs were compared. Thirty-eight patients with 2 or more episodes of SCHs (mean age, 68.2±8.9 years) underwent surgery for CCh. The effectiveness of surgery was evaluated by comparing the frequency of SCH preoperatively and postoperatively. RESULTS: Patients with three or more recurrent SCHs showed a significantly (P=0.003) higher grade of CCh and tended to be engaged in activities that require visual concentration, such as watching a visual display terminal, knitting, reading, and driving. More than 80% of eyes that underwent surgery to CCh showed no recurrence of the hemorrhages, and the frequency of SCH significantly (P<0.0001) decreased postoperatively. CONCLUSIONS: Moderate or severe CCh and activities that may cause dry eye can be considered to be risk factors for recurrent SCHs. Surgery to treat CCh is a useful option for patients with frequent recurrences of SCHs.

Clinical assessment of arterial stiffness with cardio-ankle vascular index: theory and applications.

Arterial stiffness is often assessed in clinical medicine, because it is not only an important factor in the pathophysiology of blood circulation but also a marker for the diagnosis and the prognosis of cardiovascular diseases. Many parameters have so far been proposed to quantitatively represent arterial stiffness and distensibility, such as pressure-strain elastic modulus (Ep), stiffness parameter (ß), pulse wave velocity (PWV), and vascular compliance (Cv). Among these, PWV has been most frequently applied to clinical medicine. However, this is dependent on blood pressure at the time of measurement, and therefore it is not appropriate as a parameter for the clinical evaluation of arterial stiffness, especially for the studies on hypertension. On the contrary, stiffness parameter ß is an index reflecting arterial stiffness without the influence of blood pressure. Recently, this parameter has been applied to develop a new arterial stiffness index called cardio-ankle vascular index (CAVI). Although this index is obtained from the PWV between the heart and the ankle, it is essentially similar to the stiffness parameter ß, and therefore it does not depend on blood pressure changes during the measurements. CAVI is being extensively used in clinical medicine as a measure for the evaluation of cardiovascular diseases and risk factors related to arteriosclerosis. In the present article, we will explain the theoretical background of stiffness parameter ß and the process to obtain CAVI. And then, the clinical utility of CAVI will be overviewed by reference to recent studies.

Stiffness of cancer cells measured with an AFM indentation method.

The stiffness of cancer cells and its changes during metastasis are very important for understanding the pathophysiology of cancer cells and the mechanisms of metastasis of cancer. As the first step of the studies on the mechanics of cancer cells during metastasis, we determined the elasticity and stiffness of cancer cells with an indentation method using an atomic force microscope (AFM), and compared with those of normal cells. In most of the past AFM studies, Young׳s elastic moduli of cells have been calculated from force-indentation data using Hertzian model. As this model is based on several important assumptions including infinitesimal strain and Hooke׳s linear stress-strain law, in the exact sense it cannot be applied to cells that deform very largely and nonlinearly. To overcome this problem, we previously proposed an equation F=a[exp(bδ)-1] to describe relations between force (F) and indentation (δ), where a and b are parameters relating with cellular stiffness. In the present study, we applied this method to cancer cells instead of Young׳s elastic modulus. The conclusions obtained are: 1) AFM indentation test data of cancer cells can be very well described by the above equation, 2) cancer cells are softer than normal cells, and 3) there are no significant locational differences in the stiffness of cancer cells between the central and the peripheral regions. These methods and results are useful for studying the mechanics of cancer cells and the mechanisms of metastasis.

Suppressor of cytokine signaling 1 (SOCS1) mitigates anterior uveitis and confers protection against ocular HSV-1 infection.

Immunological responses to pathogens are stringently regulated in the eye to prevent excessive inflammation that damage ocular tissues and compromise vision. Suppressors of cytokine signaling (SOCS) regulate intensity/duration of inflammatory responses. We have used SOCS1-deficient mice and retina-specific SOCS1 transgenic rats to investigate roles of SOCS1 in ocular herpes simplex virus (HSV-1) infection and non-infectious uveitis. We also genetically engineered cell-penetrating SOCS proteins (membrane-translocating sequence (MTS)-SOCS1, MTS-SOCS3) and examined whether they can be used to inhibit inflammatory cytokines. Overexpression of SOCS1 in transgenic rat eyes attenuated ocular HSV-1 infection while SOCS1-deficient mice developed severe non-infectious anterior uveitis, suggesting that SOCS1 may contribute to mechanism of ocular immune privilege by regulating trafficking of inflammatory cells into ocular tissues. Furthermore, MTS-SOCS1 inhibited IFN-γ-induced signal transducers and activators of transcription 1 (STAT1) activation by macrophages while MTS-SOCS3 suppressed expansion of pathogenic Th17 cells that mediate uveitis, indicating that MTS-SOCS proteins maybe used to treat ocular inflammatory diseases of infectious or autoimmune etiology.

Who pays the toll for solving the enigma of corneal herpes?

In herpetic stromal keratitis (HSK), herpes simplex virus type-1 DNA fragments and herpes simplex virus-immunoglobulin G immune complexes are present in corneas long after the infective virus has disappeared. These viral components are highly immunogenic and potentiate the production of proinflammatory cytokines and chemokines via Toll-like receptors expressed on corneal cells and macrophages. In addition, angiogenic factors, such as the vascular endothelium growth factor and the tissue-damaging enzyme, matrix metalloproteinase 9, are induced by corneal cells and macrophages through the recognition of these viral components in the pathogenesis of HSK. Upon neovascularization, robust infiltration of leukocytes via leaky new vessels is elicited. Activated polymorphonuclear leukocytes (PMNs) secrete hydrogen peroxide and myeloperoxidase, which inhibit viral growth. PMNs also produce tumor necrosis factor, monokine-induced by interferon-γ (CXCL9), and nitric oxide. These factors provide a local environment that can induce the differentiation of peripheral CD4* T cells to induce Th1-predominant immunopathology. Thus, strategies developed to alter these pathways should lead to new preventative and therapeutic measures for the treatment of HSK.

Biomechanical study of healing of patellar tendon after resection of the central one-third in an adult-mature rabbit model.

The present study was performed to investigate effects of ageing on biomechanical properties of healing tissues of the patellar tendon (PT) after the removal of its central portion. An entire one-third defect was made in the PT of 0.5 year- (0.5 yr) and 2 year-old rabbits (2 yr). After 6 or 12 weeks, the tissue regenerated in the defect and the remaining, residual tissue was examined biomechanically and histologically. Age-related difference in the PT length was only observed in operated tendons at 6 weeks, and in the cross-sectional area such difference was observed only in unoperated, normal tendons. At 12 weeks, tensile strength and tangent modulus of regenerated tissues in 0.5 yr were significantly higher than those in 2 yr. By contrast, these properties of residual tissues in 2 yr were significantly higher than those of 0.5 yr at 6 weeks. The histology of each of regenerated and residual tissues was essentially similar between the two age groups. Accordingly, ageing exhibited adverse effects on the mechanical properties of tissues regenerated in the central third defect of the PT. This may be due to high tangent modulus of normal and residual PT tissues in aged animals, which reduces the amount of mechanical stimulation applied to regenerated tissues during healing.

Inhibition of HSV-1 by chemoattracted neutrophils: supernatants of corneal epithelial cells (HCE) and macrophages (THP-1) treated with virus components chemoattract neutrophils (PMN), and supernatants of PMN treated with these conditioned media inhibit viral growth.

The role of PMNs (neutrophils) in corneal herpes was studied using an in vitro system. Human corneal cells (HCE) and macrophages (THP-1) infected with HSV-1 or treated with virus components (DNA or virus immune complexes) released chemokines, which attracted PMNs. Highly reactive oxygen species were detected in PMNs. PMNs inhibited HSV when overlaid onto infected HCE cells (50:1). PMNs incubated with the supernatants of HCE cells treated with virus components released H(2)O(2) and myeloperoxidase. These inhibited virus growth. PMNs released NO and MIG, which may differentiate CD4 T cells to Th1. PMNs participate in innate immune responses, limit virus growth, and initiate immunopathology.

Effects of long-term feeding of α-glucosylhesperidin on the mechanical properties of rabbit femoral arteries.

Many people are sensitive to cold, resulting in poor blood circulation. There is evidence that hesperidin results in increased peripheral circulation and skin temperature. A transglycosylated hesperidin, α-glucosylhesperidin, is more bioabsorbable than hesperidin. In the present study, biomechanical studies were performed on the effects of long-term feeding of α-glucosylhesperidin on the contractile response (diameter response) and stiffness of femoral arteries excised from rabbits. Animals in the normal (non-treated), low, and high groups were fed 0, 150 and 4500 mg/day, respectively, of α-glucosylhesperidin for about 24 weeks. The feeding of α-glucosylhesperidin did not change arterial stiffness nor mean blood flow rate in the femoral artery; however, it increased mean aortic blood pressure and decreased arterial diameter at 100 mmHg in the high group. The diameter responses developed by 10-5 M of norepinephrine were significantly lower in the high and low groups than in non-treated group. This result indicates that, due to the long-term feeding of α-glucosylhesperidin, arterial contraction induced by the neurotransmitter of sympathetic nerves decreases. It was estimated that blood flow in such muscular arteries as the femoral artery is maintained at normal by α-glucosylhesperidin even under the conditions of autonomic imbalance and cold intolerance.

Role of human herpes virus 6 in corneal inflammation alone or with human herpesviruses.

PURPOSE: : The purpose of this study was to determine the association of human herpes virus 6 (HHV-6) and/or other human herpesviruses in corneal inflammation using polymerase chain reaction (PCR). METHODS: : We collected tear films, conjunctival smears, and a corneal button of inflamed cornea, and the presence of HHV-6 and other herpesviruses in these samples were assessed by a nested PCR. RESULTS: : In tear films collected from 3 of 9 patients with dendritic keratitis, HHV-6 DNA was positive twice, together with herpes simplex virus (HSV) or varicella zoster virus DNA most often, during the acute phase of the disease. Two other patients in this group were either positive for HSV-1 and varicella zoster virus or for HSV-1 and Epstein-Barr virus DNA but negative for HHV-6. When another 12 patients' smear samples from corneal ulcer or keratouveitis were examined, 9 were positive for HHV-6 DNA. Of these, 4 were positive for HSV-1 simultaneously, whereas the remaining 5 patients were negative for HSV-1. One patient's smear was positive for HSV-1 but not for HHV-6. In the corneal button, both HSV and HHV-6 DNAs were positive by nested PCR. HHV-6 was also positive by nested PCR in the conjunctival swab obtained from the contralateral inflamed eye of the patient. CONCLUSIONS: : In 22 patients with corneal inflammation, HHV-6 was positive in 14 of 22 patients and HSV-1 was found in 9 of those patients. These data indicated that the association of HHV-6 with disease was more frequent than with other herpesviruses and that HHV-6 may be another sole causative agent for corneal inflammation.

Effects of maturation on the mechanical properties of regenerated and residual tissues in the rabbit patellar tendon after resection of its central one-third.

BACKGROUND: The central one-third portion of the patellar tendon is commonly used as a graft for the reconstruction of the anterior cruciate ligament. Although several studies have been carried out on mechanical properties of healing tendons in mature animals, there have been no studies on regenerated and residual tissues in the immature patellar tendon after the removal of its central portion. METHODS: An entire one-third defect was made in the patellar tendon of 2-, 3- and 6-month-old rabbits. After 3 weeks, the tissue regenerated in the defect and the residual tissue were biomechanically and histologically evaluated. FINDINGS: The length of patellar tendons in 6-month-old animals after the resection of its central one-third was significantly longer than that in age-matched controls. The cross-sectional area of all operated tendons was significantly larger compared to age-matched controls. There were no significant effects of maturation on the mechanical properties of regenerated and residual tissues in operated tendons, although tensile strength and tangent modulus of normal tendons were significantly greater in 6-month rabbits than in immature ones. The histology of each of regenerated and residual tissues was similar in the three groups. INTERPRETATION: There were no remarkable effects of maturation on regenerated and residual tissues after the removal of the central one-third tendon. However, the strength and the modulus of normal tendons are significantly lower in immature patients than in mature ones. Therefore, surgeons should take account of the inferior mechanical properties of the tendon in skeletally immature patients at the time of surgeries for the reconstruction of the anterior cruciate ligament.

Adaptation and remodeling of vascular wall; biomechanical response to hypertension.

Living organs, tissues, and cells functionally adapt themselves to mechanical demands, and remodel by changing geometry, structure, and properties. The key factor for this phenomenon is "Mechanical Stress". Major stresses applied to blood vessels inside the body are: (1) hoop stress induced by blood pressure, that is normal stress in the wall circumferential direction, (2) wall shear stress developed by blood flow, and (3) axial stress by elongation in the axial direction. This review article deals with biomechanical studies on the responses of arterial and venous wall to the elevation of blood pressure. One of the specific biomechanical manifestations to arterial wall adaptation in response to hypertension is wall hypertrophy. This restores circumferential wall stress, i.e. hoop stress, at in vivo operating pressure to a normal value, and changes arterial stiffness to an optimal level. Vascular smooth muscle cells are activated by hypertension. Essentially similar phenomena are also observed in venous wall.

Effects of stress shielding and subsequent restressing on mechanical properties of regenerated and residual tissues in rabbit patellar tendon after resection of its central one-third.

Central third of patellar tendon (PT) is used as an autograft for anterior cruciate ligament (ACL) reconstruction. Previous studies investigated temporal changes in material properties of healing tissues in PT after resection of the central third. However, no study has been performed on effects of stress shielding (SS) and restressing (RS) on the properties of healing tissues. The present study hypothesised that SS adversely affects the mechanical integrity of healing tissues, which is recovered by subsequent RS. An entire rectangular defect was created in the central third of rabbit PT. Operated PTs were subjected to either SS or no stress shielding (NSS). A subgroup of stress-shielded PTs was followed by the resumption of normal loading, namely RS. Tensile properties of tissues regenerated in the defect and residual tendons were evaluated. Regenerated tissues of SS for 3 weeks resulted in significantly lower strength than NSS, which was recovered to NSS level by 3 weeks of RS. Strength of residual tissues in RS reversed SS effects, leading to the strength at NSS level after 12 weeks. However, tangent modulus of residual tissues in RS was still significantly lower than that of NSS at 12 weeks. Therefore, SS induces detrimental effects on the mechanical integrity of healing PTs, and the response to RS was different between regenerate and residual tissues, the latter of which took longer period to reach NSS level.

HSV immune complex (HSV-IgG: IC) and HSV-DNA elicit the production of angiogenic factor VEGF and MMP-9.

Angiogenesis and inflammatory mediators are critical pathogenic factors in herpetic stromal keratitis (HSK). Since disease progresses without infectious virus, HSV-DNA and HSV-IgG complexes (HSV-IC) may contribute to HSK by triggering these factors. Production of VEGF and MMP-9 was studied in vitro using corneal epithelial cells (HCE), fibroblasts (HCRF) and macrophages (THP-1). VEGF was elevated in HCRF and THP-1 following treatment with HSV-DNA and HSV-IC. MMP-9 was elevated in THP-1 but not in corneal cells. When anti-HSV-IgG(Fab')2 complexes stimulated THP-1, MMP-9 was reduced to control levels. Pretreatment of THP-1 with anti-TLR-2 and -3 inhibited MMP-9 production. Thus, HSV-IC may stimulate THP-1 through the Fc receptor and TLRs. Proinflammatory cytokines (IL-1b, IL-6, and TNF-alpha) increased VEGF and MMP-9 in corneal cells and macrophages. These studies indicate that the continued presence of HSV-DNA and HSV-IC contribute to angiogenesis and inflammation in HSK. Thus, cytokines and TLRs may be potential targets for intervention.