The interaction of endothelin-1 and TGF-ß1 mediates vascular cell remodeling.

BACKGROUND: Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-ß1) and connective tissue growth factor. OBJECTIVE: To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC. METHODS: PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-ß1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580. RESULTS: ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27((Kip)). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-ß1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-ß1. CONCLUSION AND CLINICAL RELEVANCE: ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-ß1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-ß1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension.

VPAC1 receptor expression in peripheral blood mononuclear cells in a human endotoxemia model.

BACKGROUND: Vasoactive intestinal peptide (VIP) exerts immune-modulatory actions mainly via VPAC1 receptor stimulation. VPAC1 may be a treatment target of inflammatory diseases, but little is known about the receptor expression profile in immune-competent cells in vivo. MATERIAL AND METHODS: 20 male healthy subjects received a single intravenous bolus of 2 ng/kg body weight Escherichia coli endotoxin (LPS). Receptor status was evaluated in peripherial blood cells before and 3, 6 and 24 h after LPS by FACS analysis and q-PCR. VIP plasma concentrations were measured by ELISA. RESULTS: Granulocytes accounted for 51% of leukocytes at baseline and 58 ± 37% were positive for VPAC1. The granulocyte population increased 2.6 fold after LPS, and a transient down-regulation of VPAC1 to 28 ± 23% was noted at 3 h (p < 0.001), which returned to baseline at 24 hours. Baseline VPAC1 expression was low in lymphocytes (6.3 ± 3.2%) and monocytes (11 ± 9.6%). In these cells, LPS up-regulated VPAC1 at 6 h (13.2 ± 4.9%, p < 0.001) and 24 h (31.6 ± 20.5%, p = 0.001), respectively. Consistent changes were noted for the VIP-receptors VPAC2 and PAC1. VPAC1, VPAC2 and PAC1 mRNA levels were unchanged in peripheral blood mononuclear cells (PBMC). VIP plasma concentration increased from 0.5 ± 0.3 ng/ml to 0.7 ± 0.4 ng/ml at 6 h after LPS (p < 0.05) and returned to baseline within 24 h. CONCLUSION: The time profile of VPAC receptor expression differs in granulocytes, monocytes and lymphocytes after LPS challenge in humans. Changes in circulating VIP concentrations may reflect innate immune responses.

An exploratory microdialysis study investigating the effect of repeated application of a diclofenac epolamine medicated plaster on prostaglandin concentrations in skeletal muscle after standardized physical exercise.

AIM: Muscle injuries and extensive exercise are associated with cyclo-oxygenase dependent formation of inflammatory prostaglandins. Since the effect of topical administration of non-steroidal anti-inflammatory drugs (NSAIDs) on local cyclo-oxygenase is unknown, the present exploratory, open label, non-randomized study set out to measure exercise induced release of prostaglandins before and after epicutaneous administration of diclofenac. METHODS: Microdialysis was used to determine the local interstitial concentration of PGE2 and 8-iso-PGF2α as well as diclofenac concentrations in the vastus lateralis under rest, dynamic exercise and during recovery in 12 healthy subjects at baseline and after a treatment phase applying a total of seven plasters medicated with 180 mg of diclofenac epolamine over 4 days. RESULTS: At baseline PGE2 concentrations were 1169 ± 780 pg ml(-1) at rest and 1287 ± 459 pg ml(-1) during dynamic exercise and increased to 2005 ± 1126 pg ml(-1) during recovery. After treatment average PGE2 concentrations were 997 ± 588 pg ml(-1) at rest and 1339 ± 892 pg ml(-1) during exercise. In contrast with the baseline phase no increase in PGE2 concentrations was recorded during the recovery period after treatment (PGE2 1134 ± 874 pg ml(-1)). 8-iso-PGF2α was neither affected by exercise nor by treatment with diclofenac. Local and systemic concentrations of diclofenac were highly variable but comparable with previous clinical pharmacokinetic studies. CONCLUSIONS: We can hypothesize an effect of topical diclofenac epolamine plaster on limiting the increase of local concentrations of the pro-inflammatory prostaglandin PGE2 induced in the muscle of healthy human subjects following standardized physical exercise. No effect of diclofenac treatment on 8-iso-PGF2α concentrations was observed, mainly since isoprostane is produced by a free radical-catalyzed lipid peroxidation mechanism independent of cyclo-oxygenases.

Tissue pharmacokinetics of ertapenem at steady-state in diabetic patients with leg infections.

OBJECTIVES: Ertapenem pharmacokinetics were determined in the interstitium of healthy tissue and of infected tissue of patients suffering from diabetic foot infections, to evaluate if antibiotic concentrations at the target site are sufficient to achieve bacterial killing. PATIENTS AND METHODS: Nine patients with diabetic foot infections received 1 g of ertapenem per day intravenously. At steady-state, ertapenem concentrations were measured over 8 h in plasma and in the interstitium of healthy subcutaneous adipose tissue and of soft tissue adjacent to the foot infection using microdialysis. RESULTS: The maximum total concentration (Cmax) of ertapenem in plasma was 59.4 ± 12.9 mg/L. Free interstitial Cmax in the infected leg (4.5 ± 2.7 mg/L) was significantly higher (P=0.01) than in healthy subcutaneous tissue (2.4 ± 1.6 mg/L). For bacterial pathogens with an MIC of 1 mg/L, the free mean 'time above MIC' (T>MIC) in the interstitium of infected tissue was calculated to be 38%± 25% of the 24 h dosing interval. Accordingly, bacteriostatic (T>MIC >20%) and maximal bactericidal (T>MIC >40%) effects would be reached in 8/9 and 4/9 diabetic feet, respectively. CONCLUSIONS: Although total plasma concentrations of ertapenem were lower in diabetics than reported for healthy subjects, free interstitial tissue concentrations in diabetics were similar to those known from healthy volunteers. Penetration of ertapenem into the interstitium of inflamed tissue of diabetic feet was not impaired in spite of angiopathy. Daily doses of >1 g of ertapenem might be considered to optimize bactericidal effects in diabetic foot infections caused by moderately susceptible strains.

Clinical potential of VIP by modified pharmaco-kinetics and delivery mechanisms.

Vasoactive intestinal peptide (VIP) conveys various physiological effects in the digestive tract, nervous and cardiovascular system, airways, reproductive system, endocrine system, and more. A family of specific membrane bound receptors, termed VPAC1, VPAC2, and PAC1, bind VIP and trigger the effects. Many of them are of clinical interest. To date more than two thousand publications suggest the use of VIP in diseases like asthma, erectile dysfunction, blood pressure regulation, inflammation, endocrinology, tumours, etc. Despite this considerable potential, the peptide is not regularly used in clinical settings. A key problem is the short half life of inhaled or systemically administered VIP due to rapid enzymatic degradation. This shortcomings could be overcome with stable derivates or improved pharmacokinetics. A promising strategy is to use biocompatible and degradable depots, to protect the peptide from early degradation and allow for controlled release. This review focuses on aspects of clinical applications of VIP and the idea to use formulations based on biodegradable particles, to constitute a dispersible VIP-depot. Smart particle systems protect the peptide from early degradation, and assist the sustainable cell targeting with VIP for therapeutic or imaging purposes.

Penetration of doripenem into skeletal muscle and subcutaneous adipose tissue in healthy volunteers.

Sufficient antibiotic concentrations at the infection site are a prerequisite for good bacterial killing. This study was performed to determine pharmacokinetics of doripenem in soft tissues and saliva. Six healthy male volunteers received a single intravenous infusion of 500 mg doripenem over 1 h. The concentrations of doripenem were measured over 8 h in saliva, plasma, and extracellular space fluid of skeletal muscle and subcutaneous adipose tissue employing in vivo microdialysis. Unbound drug concentrations were determined using ultra-high-performance liquid chromatography-tandem mass spectrometry. Maximum concentrations of doripenem were 15.3 ± 6.0 mg/liter in plasma, 9.9 ± 2.3 mg/liter in subcutaneous adipose tissue, 6.6 ± 2.9 mg/liter in skeletal muscle, and 0.5 ± 0.2 mg/liter in saliva. Areas under the concentration-time curve (AUC) from 0 to infinity were 26.3 ± 10.1, 20.4 ± 3.8, 12.8 ± 3.0, and 1.0 ± 0.5 mg · h/liter in plasma, adipose tissue, skeletal muscle, and saliva, respectively. Ratios of AUC in adipose tissue, skeletal muscle, and saliva to those in plasma were 0.84 ± 0.28, 0.53 ± 0.19, and 0.04 ± 0.03, respectively. In all six volunteers, a threshold of ≥40% for "time above MIC," an index indicative of good antimicrobial activity, was exceeded in adipose tissue for MICs of ≤2 mg/liter and in skeletal muscle for MICs ≤1.5 mg/liter. Doripenem penetrates well into interstitial space fluid of skeletal muscle and adipose tissue, suggesting good antimicrobial activity in infected soft tissues, whereas it is detectable in relatively low concentrations in saliva.

Vasoactive intestinal peptide (VIP) receptor expression in monocyte-derived macrophages from COPD patients.

Vasoactive intestinal peptide (VIP) is one of the most abundant molecules found in the respiratory tract. Due to its anti-inflammatory and bronchodilatatory properties, it has been proposed as a novel treatment for chronic obstructive pulmonary disease (COPD). The actions of VIP are mediated via three different G-protein-coupled receptors (VPAC1, VPAC2 and PAC1) which are expressed in the respiratory tract and on immunocompetent cells including macrophages. Alveolar macrophages (AM) are key players in the pathogenesis of COPD and contribute to the severity and progression of the disease. While VPAC1 has been reported to be elevated in subepithelial cells in smokers with chronic bronchitis, little is known about VPAC expression of AM in COPD patients. AM from COPD patients show a strong VPAC1 expression which exceeds VPAC2. A similar receptor expression pattern was also observed in lipopolysaccharide (LPS)-activated monocyte-derived macrophages (MDM) from healthy volunteers and COPD patients. VIP has been shown to down-regulate interleukin 8 (IL-8) secretion significantly in MDM after LPS stimulation. The response to VIP was similar in MDM from COPD patients and healthy volunteers. Our results indicate that VPAC1 up-regulation in macrophages is a common mechanism in response to acute and chronic pro-inflammatory stimuli. Although VPAC1 up-regulation is dominant, both receptor subtypes are necessary for optimal anti-inflammatory signaling. The high VPAC1 expression in AM may reflect the chronic pro-inflammatory environment found in the lung of COPD patients. Treatment with VIP may help to decrease the chronic inflammation in the lung of COPD patients.

Abnormal pulmonary arterial pressure limits exercise capacity in patients with COPD.

OBJECTIVE: Pulmonary hypertension (PH) is common in patients with chronic obstructive pulmonary disease (COPD). Mean pulmonary artery pressure (mPAP) is often only slightly elevated at rest but is increased by exercise. The purpose of this study was to determine whether abnormal pulmonary artery pressure impairs exercise capacity in patients with COPD. PATIENTS AND METHODS: 42 patients with moderate-to-very-severe COPD (28 men, 14 women) underwent symptom-limited incremental cardiopulmonary exercise testing and also right-heart catheterization at rest. Abnormal pulmonary artery pressure was defined as mPAP>20 mmHg at rest. RESULTS: Resting mPAP was elevated in 32 patients (PH, mPAP=26.8+/-5.9 mmHg) and normal in 10 non-hypertensive (NPH) patients (NPH, mPAP=16.8+/-2 mmHg). There were no significant differences in lung function between the PH and NPH groups. Maximum oxygen uptake during exercise (VO2max) was significantly lower in PH (785+/-244 ml/min) than in NPH (1052+/-207 ml/min, P=0.004). Dead-space ventilation (Vd/Vt) was greater in PH (P=0.05) with higher VE/VCO2 (ratio of minute ventilation to carbon dioxide output=47.3+/-10 vs 38.6+/-3.5, P=0.025) and significantly higher arterial-end-tidal pCO2 difference [p(a-ET)CO2]. Pulmonary vascular resistance measured at rest correlated significantly with VO2max, VE/VCO2 and p(a-ET)CO2. CONCLUSIONS: In patients with COPD, abnormal pulmonary artery pressure impairs gas exchange, decreases maximum oxygen uptake during exercise and impairs exercise capacity.

Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension.

Primary pulmonary hypertension is a fatal disease causing progressive right heart failure within 3 years after diagnosis. We describe a new concept for treatment of the disease using vasoactive intestinal peptide, a neuropeptide primarily functioning as a neurotransmitter that acts as a potent systemic and pulmonary vasodilator. Our rationale is based on the finding of a deficiency of the peptide in serum and lung tissue of patients with primary pulmonary hypertension, as evidenced by radioimmunoassay and immunohistochemistry. The relevance of this finding is underlined by an upregulation of corresponding receptor sites as shown by Northern blot analysis, Western blot analysis, and immunological techniques. Consequently, the substitution with the hormone results in substantial improvement of hemodynamic and prognostic parameters of the disease without side effects. It decreased the mean pulmonary artery pressure in our eight study patients, increased cardiac output, and mixed venous oxygen saturation. Our data provide enough proof for further investigation of vasoactive intestinal peptide and its role in primary pulmonary hypertension.