Buprenorphine-Sustained Release Alters Hemodynamic Parameters in a Rat Burn Model.

BACKGROUND: It has been previously shown that anesthesia and analgesia can affect outcomes in the rat burn model and that buprenorphine alleviated pain without drastically altering the outcomes of interest. Recently, the use of a sustained release (SR) formulation of buprenorphine has been promoted over conventional buprenorphine. In this study, we assessed whether buprenorphine-SR altered hemodynamic parameters in our rat model of severe burn injury. MATERIALS AND METHODS: Adult male Sprague-Dawley rats were randomized to receive either conventional buprenorphine (0.05 mg/kg) or buprenorphine-SR (1 mg/kg). Buprenorphine-SR was administered 24 h before the experiment. Buprenorphine was administered on the day of experiment. These groups were further randomized to control or scald burn (60% of total body surface area). Systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) were measured using a noninvasive blood pressure system before receiving analgesia and after 72 h. RESULTS: As expected, HR was significantly higher after burn injury regardless of analgesic (P <0.0001). Both SBP and DBP were significantly decreased in burned animals receiving conventional buprenorphine (P < 0.0001), but neither was altered in the buprenorphine-SR-treated burned animals. However, SBP, DBP, and HR were significantly increased after 72 h in control animals receiving buprenorphine-SR (P < 0.0001). CONCLUSIONS: These data indicate that buprenorphine-SR alters the hemodynamic response to injury and may not be an appropriate choice for a model of severe burn injury. If this analgesic is used, investigators must cautiously form conclusions, especially in experimental conditions that would be expected to alter cardiac hemodynamics.

The Effect of Burn Trauma on Lipid and Glucose Metabolism: Implications for Insulin Sensitivity.

Severe burns represent a unique form of trauma in terms of the magnitude and persistence of the stress response they incur. Given advances in acute burn care in the last quarter of a century and the resultant reduction in mortality rates, even for those with massive burns, greater emphasis is now placed on understanding the metabolic stress response to severe burn trauma in order to devise strategies that promote recovery and reduce morbidity. Derangements in metabolism including protein and lipid redistribution and altered glucose handling are hallmarks of the pathophysiological response to burn trauma. In this review article, we aim to distill and discuss the c urrent literature concerning the effect of burn trauma on lipid and glucose metabolism. Furthermore, we will discuss the implications of altered lipid metabolism with regards to insulin sensitivity and glucose control, while discussing the utility of agents and strategies aimed at restoring normal lipid and glucose metabolism in burned patients.

Biventricular differences in ß-adrenergic receptor signaling following burn injury.

Burn injury detrimentally affects the myocardium, primarily due to over-activation of ß-adrenergic receptors (ß-AR). Autopsy reports from our institution reveal that patients often suffer from right ventricle (RV) failure. Since burn injury affects ß-AR signaling in the left ventricle (LV), we proposed that ß-AR signaling may also be altered in the RV. A rodent model with a scald burn of 60% of the total body surface area was used to test this hypothesis. Ventricles were isolated 7 days post-burn. We examined the expression of ß-ARs via Western blotting and the mRNA expression of downstream signaling proteins via qRT-PCR. Cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activity were measured in membrane and cytosolic fractions, respectively, using enzyme immunoassay kits. ß1-AR protein expression was significantly increased in the RV following burn injury compared to non-burned RV but not in the LV (p = 0.0022). In contrast, ß2-AR expression was unaltered among the groups while Gαi expression was significantly higher in the LV post-burn (p = 0.023). B-arrestin-1 and G-protein coupled receptor kinase-2 mRNA expression were significantly increased in the left ventricle post-burn (p = 0.001, p<0.0001, respectively). cAMP production and PKA activity were significantly lower in the LV post-burn (p = 0.0063, 0.0042, respectively). These data indicate that burn injury affects the ß-AR signaling pathway in the RV independently of the LV. Additionally, non-canonical ß-AR signaling may be activated in the RV as cAMP production and PKA activity were unchanged despite changes in ß1-AR protein expression.

Safety of Nebulized Epinephrine in Smoke Inhalation Injury.

This pilot study was conducted to profile safety of nebulized racemic epinephrine when used as a therapy for smoke inhalation injury in severely burned children. We enrolled 16 patients who were 7 to 19 years of age ([mean ± SD], 12 ± 4 years) with burns covering more than 30% of the TBSA (55 ± 17%) and smoke inhalation injury, as diagnosed by bronchoscopy at burn center admission. Patients were randomized to receive either standard of care (n = 8), which consisted of nebulized acetylcysteine, nebulized heparin, and nebulized albuterol, or to receive standard of care plus nebulized epinephrine (n = 8). Primary endpoints were death, chest pain, and adverse changes in cardiopulmonary hemodynamics (arrhythmia, arterial blood pressure, electrocardiographic [ST segment] changes, and peak inspiratory pressure). Additional endpoints included total days on ventilator, pulmonary function, and physiological cardiopulmonary measurements at intensive care unit discharge. No adverse events were observed during or after the nebulization of epinephrine, and no deaths were reported that were attributable to the administration of nebulized epinephrine. The groups did not significantly differ with regard to age, sex, burn size, days on ventilator, pulmonary function, or cardiopulmonary fitness. Results of this pilot trial indicate epinephrine to be safe when administered to pediatric burn patients with smoke inhalation injury. Current data warrant future efficacy studies with a greater number of patients.

Herpesviradae infections in severely burned children.

OBJECTIVE: Burn-related immunosuppression can promote human herpesviridae infections. However, the effect of these infections on morbidity and mortality after pediatric burn injuries is unclear. METHODS: We retrospectively analyzed pediatric patients with burns ≥10% of the total body surface area (TBSA) who were admitted between 2010 and 2015. On clinical suspicion of a viral infection, antiviral therapy was initiated. Viral infection was confirmed via Tzanck smear, viral culture, and/or PCR. Study endpoints were mortality, days of antiviral agent administration, type of viral test used, type of viral infection, and length of hospitalization. RESULTS: Of the 613 patients were analyzed, 28 presented with clinically diagnosed viral infections. The use of Tzanck smears decreased over the past 5 years, whereas PCR and viral cultures have become standard. Patients with viral infections had significantly larger burns (53±15% vs. 38±18%, p<0.001); however, length of stay per TBSA burn was comparable (0.5±0.4 vs. 0.6±0.2, p=0.211). The most commonly detected herpesviridae was herpes simplex virus 1. Two patients died due to sepsis, which was accompanied by HSV infection. The mortality rate among all patients (2.7%) was comparable to that in the infected group (7.1%, p=0.898). Acyclovir was given systemically for 9±8days (N=76) and/or topically for 9±9days for HSV (N=39, combination of both N=33). Ganciclovir was prescribed in three cases for CMV. CONCLUSIONS: Viral infections occur more commonly in patients suffering from larger burns, and HSV infections can contribute to mortality.

Long-term effect of critical illness after severe paediatric burn injury on cardiac function in adolescent survivors: an observational study.

BACKGROUND: Sepsis, trauma, and burn injury acutely depress systolic and diastolic cardiac function; data on long-term cardiac sequelae of pediatric critical illness are sparse. This study evaluated long-term systolic and diastolic function, myocardial fibrosis, and exercise tolerance in survivors of severe pediatric burn injury. METHODS: Subjects at least 5 years after severe burn (post-burn:PB) and age-matched healthy controls (HC) underwent echocardiography to quantify systolic function (ejection fraction[EF%]), diastolic function (E/e'), and myocardial fibrosis (calibrated integrated backscatter) of the left ventricle. Exercise tolerance was quantified by oxygen consumption (VO2) and heart rate at rest and peak exercise. Demographic information, clinical data, and biomarker expression were used to predict long-term cardiac dysfunction and fibrosis. FINDINGS: Sixty-five subjects (PB:40;HC:25) were evaluated. At study date, PB subjects were 19±5 years, were at 12±4 years postburn, and had burns over 59±19% of total body surface area, sustained at 8±5 years of age. The PB group had lower EF% (PB:52±9%;HC:61±6%; p=0.004), E/e' (PB:9.8±2.9;HC: 5.4±0.9;p<0.0001), VO2peak (PB:37.9±12;HC: 46±8.32 ml/min/kg; p=0.029), and peak heart rate (PB:161±26;HC:182±13bpm;p=0.007). The PB group had moderate (28%) or severe (15%) systolic dysfunction, moderate (50%) or severe diastolic dysfunction (21%), and myocardial fibrosis (18%). Biomarkers and clinical parameters predicted myocardial fibrosis, systolic dysfunction, and diastolic dysfunction. INTERPRETATION: Severe pediatric burn injury may have lasting impact on cardiac function into young adulthood and is associated with myocardial fibrosis and reduced exercise tolerance. Given the strong predictive value of systolic and diastolic dysfunction, these patients might be at increased risk for early heart failure, associated morbidity, and mortality. FUNDING: Conflicts of Interest and Sources of Funding: The authors do not have any conflicts of interest to declare. This work was supported by NIH (P50 GM060338, R01 GM056687, R01 HD049471, R01 GM112936, R01-GM56687 and T32 GM008256), NIDILRR (H133A120091, 90DP00430100), Shriners Hospitals for Children (84080, 79141, 79135, 71009, 80100, 71008, 87300 and 71000), FAER (MRTG CON14876), and the Department of Defense (W81XWH-14-2-0162 and W81XWH1420162). It was also made possible with the support of UTMB's Institute for Translational Sciences, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advancing Translational Sciences (NIH).

Effects of different duration exercise programs in children with severe burns.

INTRODUCTION: Burns lead to persistent and detrimental muscle breakdown and weakness. Standard treatment at our institution includes a voluntary 12-week rehabilitative exercise program to limit and reverse the effects of increased muscle catabolism. In the present work, we investigated if different durations of exercise, 6 or 12 weeks, produce comparable improvements in muscle strength, body composition, and cardiopulmonary fitness. METHODS: We prospectively enrolled and randomized patients with ≥30% total body surface area (TBSA) burned to receive 6 or 12 weeks of exercise rehabilitation. Patients were evaluated for muscle strength, oxygen consumption capacity, and lean body mass at discharge (n=42) and after exercise. After 6 weeks (n=18) or 12 weeks (n=24) of exercise training, leg muscle strength was assessed as peak torque per body weight using a Biodex isokinetic dynamometer. Oxygen consumption capacity, measured as peak VO2, was studied using a standard treadmill-based test, and lean body mass was determined using dual-energy X-ray absorptiometry. RESULTS: Significant improvements in muscle strength, peak VO2, and lean body mass were seen after 6 weeks of exercise training (p<0.001), with only significant improvements in peak VO2 being seen after 6 weeks more of training. CONCLUSION: These data suggest that a 6-week rehabilitative exercise program is sufficient for improving muscle strength, body composition, and cardiopulmonary fitness in pediatric burn patients. However, continuation of at- or near-home cardiopulmonary training following the 6 weeks of at-hospital rehabilitation may be useful.

Human herpes viruses in burn patients: A systematic review.

OBJECTIVE: The contribution of human herpes viruses, including herpes simplex virus (HSV), cytomegalovirus (CMV), and varicella zoster virus (VZV) to morbidity and mortality after burns remains controversial. This systematic review was undertaken to assess evidence of herpes virus-related morbidity and mortality in burns. MATERIALS AND METHODS: PubMed, Ovid, and Web of Science were searched to identify studies of HSV, CMV, or VZV infections in burn patients. Exclusion criteria included: A level of evidence (LoE) of IV or V; nonhuman in vivo studies; and non-English articles. There was no limitation by publication date. RESULTS: Fifty articles were subjected to full-text analysis. Of these, 18 had LoE between I-III and were included in the final review (2 LoE I, 16 LoE II-III). Eight had a prospective study design, 9 had a retrospective study design, and 1 included both. CONCLUSIONS: No direct evidence linked CMV and HSV infection with increased morbidity and mortality in burns. Following burn, CMV reactivation was more common than a primary CMV infection. Active HSV infection impaired wound healing but was not directly correlated to mortality. Infections with VZV are rare after burns but when they occur, VZV infections were associated with severe complications including mortality. The therapeutic effect of antiviral agents administered after burns warrants investigation via prospective randomized controlled trials.

Propranolol Reduces Cardiac Index But does not Adversely Affect Peripheral Perfusion in Severely Burned Children.

PURPOSE: The aim of this study was to quantify the effect of propranolol on hemodynamic parameters assessed using the PiCCO system in burned children. METHODS: We analyzed hemodynamic data from patients who were randomized to receive either propranolol (4 mg/kg/day) or placebo (control), which was initiated as a prospective randomized controlled trial. Endpoints were cardiac index (CI), percent predicted heart rate (%HR), mean arterial pressure (MAP), percent predicted stroke volume (%SV), rate pressure product (RPP), cardiac work (CW), systemic vascular resistance index (SVRI), extravascular lung water index (EVLWI), arterial blood gases, events of lactic acidosis, and mortality. Mixed multiple linear regressions were applied, and a 95% level of confidence was assumed. RESULTS: One hundred twenty-one burned children (control: n = 62, propranolol: n = 59) were analyzed. Groups were comparable in demographics, EVLWI, SVRI, %SV, arterial blood gases, Denver 2 postinjury organ failure score, incidence of lactic acidosis, or mortality. Percent predicted HR, MAP, CI, CW, and RPP were significantly reduced in the propranolol-treated group (P <0.01). CONCLUSIONS: Propranolol significantly reduces cardiogenic stress by reducing CI and MAP in children with severe burn injury. However, peripheral oxygen delivery was not reduced and events of lactic acidosis as well as organ dysfunction was not higher in propranolol treated patients.

Long-term effects of physical exercise during rehabilitation in patients with severe burns.

BACKGROUND: We have reported that a 12-week exercise program is beneficial for the exercise performance of severely burned children. It is not known, however, whether the beneficial effects remain at 2 years postburn. METHODS: Severely burned children who received no long-term anabolic drugs were consented to this Institutional Review Board-approved study. Patients chose between a voluntary exercise program (EX-group) and no exercise (NoEX-group) after discharge from the acute burn unit. Peak torque per lean leg mass, maximal oxygen consumption, and percent predicted peak heart rate were assessed. In addition, body mass index percentile and lean body mass index were recorded. Both groups were compared for up to 2 years postburn using mixed multiple analysis of variance. RESULTS: A total of 125 patients with a mean age of 12 ± 4 years were analyzed. Demographics between the EX-group (N = 82) and NoEX-group (N = 43) were comparable. In the EX-group, peak torque per lean leg mass, percent predicted peak heart rate, and maximal oxygen consumption increased significantly with exercise (P < .01). Between discharge and 12-24 months, body mass index percentile increased significantly in the EX-Group (P < .05) but did not change in the NoEX-group. There were no significant differences between groups in body mass index percentile, lean body mass index, peak torque per lean leg mass, and maximal oxygen consumption at 24 months postburn. CONCLUSION: Exercise significantly improves the physical performance of burned children. The benefits are limited to early time points, however, and greatly narrow with further recovery time. Continued participation in exercise activities or a maintenance exercise program is recommended for exercise-induced adaptations to continue.

Anatomy of the Vascularized Lateral Femoral Condyle Flap.

BACKGROUND: The perforators of the free lateral femoral condyle flap have yet to be adequately described in humans. Therefore, the authors investigated the perforator vessels of the lateral femoral condyle flap in an anatomical study and discussed potential clinical applications with a particular interest in its indication with vascularized bone and/or iliotibial band for tendon repair surgery. METHODS: The authors dissected thighs of 28 cadavers to evaluate the anatomical properties of perforator vessels that branch from the superior lateral genicular artery and supply bone, cartilage, subcutaneous tissue, and the iliotibial band of the lateral femoral condyle. RESULTS: In each dissected thigh, the superior lateral genicular artery was present and the average pedicle length was 38 ± 10 mm. The average diameter of the superior lateral genicular artery, proximal to its distribution into the deep articular and superficial patellar branches, was 2 ± 0.5 mm. A communication between deep articular and superficial patellar branches was seen in 96 percent of the dissected thighs. In 24 cases (86 percent), the authors were able to show the iliotibial band perforating vessel and harvest a free lateral femoral condyle flap as an osteochondral fasciocutaneous bone flap with vascularized tendon. CONCLUSIONS: Altogether, the authors' results indicate that the blood supply of the lateral femoral condyle flap is consistent and the lateral femoral condyle flap could serve as a free composite flap for complex indications in hand or limb reconstructive surgery. Clinical studies to compare the lateral femoral condyle to other well-established microsurgical free flaps are warranted.

The use of skin substitutes and burn care-a survey.

OBJECTIVE: The aim of our Internet survey was to assess the preferences of burn specialists who use skin substitutes in patients with burns covering 20% or more of their total body surface area (TBSA). METHODS: An open, voluntary Internet-based cross-sectional survey was performed. Responses to 19 noncompulsory questions, and participant career and location information were collected. RESULTS: One hundred eleven specialists from 36 countries responded to our questionnaire. Sixty participants were located in Europe (54%), followed by 31 (28%) in North America, 15 (14%) in Asia, three (3%) in South America, one (1%) in Africa, and one (1%) in Australia. The importance of skin substitutes in medium-sized burns (covering 20%-60% TBSA) was rated as "essential" by 28% and "desirable" by 56% of the respondents. In severe burns >60% of TBSA, 81% of responders rated the use of skin substitutes as "essential" and 14% as "desirable". Skin substitutes were used in daily clinical practice by 96% of all participants. Biological and synthetic dressings were used by 53%. A majority (86%) think that biological dressings do not pose a risk to patients. Allografts represent the most frequently used wound coverage (51%), followed by xenografts (28%). All participants of the survey indicated that as of yet, there is no ideal skin substitute available. CONCLUSIONS: Split-thickness autografts still represent the most used wound cover for definitive treatment of severe burns. However, creation and implementation of an ideal skin substitute have yet to be achieved and therefore should be the focus of future work.

Cardiovascular Dysfunction Following Burn Injury: What We Have Learned from Rat and Mouse Models.

Severe burn profoundly affects organs both proximal and distal to the actual burn site. Cardiovascular dysfunction is a well-documented phenomenon that increases morbidity and mortality following a massive thermal trauma. Beginning immediately post-burn, during the ebb phase, cardiac function is severely depressed. By 48 h post-injury, cardiac function rebounds and the post-burn myocardium becomes tachycardic and hyperinflammatory. While current clinical trials are investigating a variety of drugs targeted at reducing aspects of the post-burn hypermetabolic response such as heart rate and cardiac work, there is still a paucity of knowledge regarding the underlying mechanisms that induce cardiac dysfunction in the severely burned. There are many animal models of burn injury, from rodents, to sheep or swine, but the majority of burn related cardiovascular investigations have occurred in rat and mouse models. This literature review consolidates the data supporting the prevalent role that ß-adrenergic receptors play in mediating post-burn cardiac dysfunction and the idea that pharmacological modulation of this receptor family is a viable therapeutic target for resolving burn-induced cardiac deficits.

FIVE-YEAR OUTCOMES AFTER LONG-TERM OXANDROLONE ADMINISTRATION IN SEVERELY BURNED CHILDREN: A RANDOMIZED CLINICAL TRIAL.

Administration of oxandrolone, a nonaromatizable testosterone analog, to children for 12 months following severe burn injury has been shown to improve height, increase bone mineral content (BMC), reduce cardiac work, and augment muscle strength. Surprisingly, the increase in BMC persists well beyond the period of oxandrolone administration. This study was undertaken to determine if administration of oxandrolone for 2 years yields greater effects on long-term BMC and bone mineral density (BMD). Patients between 0 and 18 years of age with ≥30% of total body surface area burned were consented to an IRB-approved protocol and randomized to receive either placebo (n = 84) or 0.1 mg/kg oxandrolone orally twice daily for 24 months (n = 35). Patients were followed prospectively from the time of admission until 5 years postburn in a single-center, intent-to-treat setting. Height, weight, BMC, and BMD were recorded annually through 5 years postinjury. The long-term administration of oxandrolone for 16 ± 1 months postburn (range, 12.1-25.2 months) significantly increased whole-body (WB) BMC (p < 0.02) and lumbar spine (LS) BMC (p < 0.05); these effects were significantly pronounced for a longer time in patients who were in growth spurt years (7-18 years). When adjusted for height, sex, and age, LS BMD was found to significantly increase with long-term oxandrolone administration (p < 0.0009). Fewer patients receiving oxandrolone exhibited LS BMD z scores below -2.0 as compared with controls, indicating a significantly reduced risk for future fracture with oxandrolone administration. Long-term oxandrolone patients had significantly greater height velocity than controls throughout the first 2-year postburn (p < 0.05). No adverse side effects were attributed to the long-term administration of oxandrolone. A comparison of the current patients receiving long-term oxandrolone to previously described patients receiving 12 months of oxandrolone revealed that long-term oxandrolone administration imparted significantly greater increases in WB-BMC, WB-BMD, and LS-BMD (p < 0.05). In conclusion, the administration of oxandrolone for up to 24 months to severely burned pediatric patients significantly improves WB BMC, LS BMC, LS BMD, and height velocity. The administration of long-term oxandrolone was more efficacious than administration for 12 months. Additionally, fewer patients in the oxandrolone cohort met the diagnostic criteria for pediatric osteoporosis, pointing to a reduced risk for future bone fracture. This study demonstrates that administering oxandrolone for up to 2 years following severe burn injury results in greater improvements in BMC, BMD, and height velocity.

Impact of stress-induced diabetes on outcomes in severely burned children.

BACKGROUND: Post-burn hyperglycemia leads to graft failure, multiple organ failure, and death. A hyperinsulinemic-euglycemic clamp is used to keep serum glucose between 60 and 110 mg/dL. Because of frequent hypoglycemic episodes, a less-stringent sliding scale insulin protocol is used to maintain serum glucose levels between 80 and 160 mg/dL after elevations >180 mg/dL. STUDY DESIGN: We randomized pediatric patients with massive burns into 2 groups, patients receiving sliding scale insulin to lower blood glucose levels (n = 145) and those receiving no insulin (n = 98), to determine the differences in morbidity and mortality. Patients 0 to 18 years old with burns covering ≥ 30% of the total body surface area and not randomized to receive anabolic agents were included in this study. End points included glucose levels, infections, resting energy expenditure, lean body mass, bone mineral content, fat mass, muscle strength, and serum inflammatory cytokines, hormones, and liver enzymes. RESULTS: Maximal glucose levels occurred within 6 days of burn injury. Blood glucose levels were age dependent, with older children requiring more insulin (p < 0.05). Daily maximum and daily minimum, but not 6 am, glucose levels were significantly different based on treatment group (p < 0.05). Insulin significantly increased resting energy expenditure and improved bone mineral content (p < 0.05). Each additional wound infection increased incidence of hyperglycemia (p = 0.004). There was no mortality in patients not receiving insulin, only in patients who received insulin (p < 0.004). Muscle strength was increased in patients receiving insulin (p < 0.05). CONCLUSIONS: Burn-induced hyperglycemia develops in a subset of severely burned children. Length of stay was reduced in the no insulin group, and there were no deaths in this group. Administration of insulin positively impacted bone mineral content and muscle strength, but increased resting energy expenditure, hypoglycemic episodes, and mortality. New glucose-lowering strategies might be needed.

Reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell hyperpermeability following oxygen-glucose deprivation and reoxygenation.

OBJECTIVE: Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this. METHODS: RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively. RESULTS: OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly. CONCLUSIONS: These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.