Ansgar Berg

A novel physiological function for platelet-derived growth factor-BB in rat dermis.

1. The present experiments describe a role for platelet‐derived growth factor‐BB and cellular adhesion receptors towards extracellular matrix molecules (beta 1‐integrins) in control of interstitial fluid pressure (Pif). 2. Pif was measured in rat skin with sharpened glass capillaries (3‐7 microns) connected to a servocontrolled counter‐pressure system. 3. The collagen and laminin‐binding alpha 2 beta 1‐integrin is involved in the control of Pif since subdermal injection (5 microliters) of monoclonal hamster anti‐rat alpha 2 beta 1‐integrin IgG (anti‐alpha 2 beta 1) resulted in increased negativity of Pif. Control Pif averaged ‐0.88 +/‐ 0.23 mmHg (+/‐ S.D.) and decreased to ‐2.50 +/‐ 0.35 mmHg (P < 0.05) and ‐3.88 +/‐ 1.45 mmHg (P < 0.05) at anti‐alpha 2 beta 1 concentrations of 0.56 and 1.12 mg ml‐1, respectively. 4. The effect of anti‐alpha 2 beta 1 was abolished when platelet‐derived growth factor‐BB (PDGF‐BB) (200 ng ml‐1) was injected together with anti‐alpha 2 beta 1. 5. The time‐ and dose‐responses of PDGF‐BB to counteract increased negativity of Pif were studied further using dextran anaphylaxis as an experimental model inducing increased negativity of Pif in skin. Control Pif averaged ‐0.33 +/‐ 0.43 mmHg and fell to ‐4.10 +/‐ 1.47 mmHg within 10 min after dextran (P < 0.01). Subsequent subdermal injection of PDGF‐BB at 200 ng ml‐1 normalized Pif in 10‐20 min which became ‐1.37 +/‐ 1.23 mmHg (P < 0.01 versus dextran, P > 0.05 versus control). PDGF‐BB had little or no effect at 50 ng ml‐1. PDGF‐AA and basic fibroblast growth factor had no effect on Pif. 6. The in vivo function reported for PDGF‐BB has not been described previously and provides further evidence for active participation of connective tissue cells in control of Pif by altering tension on extracellular matrix structures.

Effect of PGE1, PGI2, and PGF2α analogs on collagen gel compaction in vitro and interstitial pressure in vivo

Acute inflammation in skin is accompanied by increased negativity of interstitial fluid pressure (PIF), which will increase capillary fluid filtration and thereby potentiate edema formation. A series of studies indicates that the connective tissue cells in rat dermis are involved in the control of PIF and mediate this response. The present study describes a novel effect of prostaglandin (PG) E1 isopropyl ester, carbaprostacyclin (PGI2 analog), and latanoprost (PGF2α analog) on edema formation and PIF in parallel with their action on the fibroblast-populated collagen gel contraction assay. The prostaglandins were injected subdermally in pentobarbital-anesthetized rats. PIF was measured with a servo-controlled counterpressure system after circulatory arrest had been induced with saturated potassium chloride. Circulatory arrest was induced to limit edema formation that would raise interstitial fluid volume and thereby attenuate a possible increased negativity of PIF. PGE1 (0.91 mM) and carbaprostacyclin (1.28 mM) lowered PIF from a control value of -0.8 ± 0.4 mmHg to -3.0 ± 0.4 ( P < 0.01) and -3.7 ± 0.9 ( P < 0.01) mmHg, respectively, within 45 min in a dose-dependent manner. Edema formation was measured in separate experiments. PGE1 and carbaprostacyclin significantly increased interstitial fluid volume (extravascular51Cr-EDTA space) at concentrations as low as 0.1 and 1.1 μM, respectively. Latanoprost had no effect on PIF or edema formation. However, latanoprost reversed, in a dose-dependent manner, an increased negativity of PIF accompanying the anaphylactic reaction to dextran. In the gel contraction assay with human diploid fibroblasts (AG 1518), a corresponding specificity was observed where PGE1 and carbaprostacyclin effectively inhibited gel contraction although latanoprost had no effect. Thus the present data demonstrate a novel effect of prostaglandins and provide further evidence for active modulation of PIF via loose connective tissue cells.

CELL INTERACTIONS WITH COLLAGEN MATRICES IN VIVO AND IN VITRO DEPEND ON PHOSPHATIDYLINOSITOL 3-KINASE AND FREE CYTOPLASMIC CALCIUM

We have investigated the role of phosphatidylinositol 3-kinase (PI3-kinase) in cellular interactions with collagenous matrices. Platelet-derived growth factor-BB (PDGF-BB) elicited a mobilization of intracellular Ca2+ in pig aortic endothelial (PAE) cells transfected with wild type PDGF beta-receptor. This response was greatly reduced in PAE cells transfected with PDGF beta-receptors mutated at positions Y740 and Y751 to prevent PI3-kinase binding. The experimental drug 1D-myo-inositol 1,2,6-trisphosphate (alpha-trinositol) induced a rapid increase and subsequent oscillations of the cytoplasmic Ca2+ concentration in cultured fibroblasts. This response was not due to an effect of alpha-trinositol on inositol 1,4,5-trisphosphate (IP3) receptors. alpha-Trinositol did not influence PDGF-BB elicited chemotaxis through collagen-coated membranes of PAE cells transfected with the wild-type PDGF beta-receptor, but restored PDGF-BB elicited chemotaxis of PAE cells transfected with the PI3-kinase binding-site mutated PDGF beta-receptor. Collagen gel contraction has been suggested to serve as a model for cellular control of interstitial fluid pressure (PIF) in dermis. The PI3-kinase inhibitors wortmannin (50 nM) and LY294002 (5 microM) inhibited the stimulation of fibroblast-mediated collagen gel contraction by 0.4 nM PDGF-BB. Injection of wortmannin in rat paw skin induced a lowering of PIF, and this effect was abolished in animals pre-treated with alpha-trinositol. Pretreatment of rats with alpha-trinositol abolished the decrease in PIF induced by injecting monoclonal anti-rat alpha 2 beta 1 integrin IgG in rat paw skin. Taken together our data indicate that cell-collagen interactions in vivo and in vitro depend on PI3-kinase, and that this dependence can be bypassed by a drug eliciting intracellular Ca2+ mobilization.

Platelet-Derived Growth Factor BB-Mediated Normalization of Dermal Interstitial Fluid Pressure After Mast Cell Degranulation Depends on β3 but Not β1 Integrins

Interstitial fluid pressure (PIF) is one of the determinants of transcapillary fluid flux and thereby interstitial fluid volume. Cell-mediated control of PIF regulates fluid content in the loose interstitial connective tissues that surround the capillary bed. To maintain a normal PIF in dermis, &bgr;1 integrins mediate the tensile strength applied by connective tissue cells on the extracellular matrix. Platelet-derived growth factor (PDGF)-BB normalizes anaphylaxis-induced reduction of PIF. Anti–&bgr;3 integrin IgG and a cyclic RGD peptide that inhibits the &agr;V&bgr;3 integrin blocked the ability of PDGF-BB to normalize the lowered PIF resulting from mast cell degranulation. PDGF-BB was unable to normalize PIF lowered as a result of mast cell degranulation in &bgr;3-negative mice. Monoclonal anti–&bgr;3 integrin IgG had no effect on PIF in normal mouse dermis. In contrast, administration of anti–&bgr;1 integrin IgM lowered PIF in normal dermis but had no effect on PDGF-BB–induced normalization of PIF after anaphylaxis. Furthermore, collagen gel contraction mediated by wild-type mouse embryonal fibroblasts were only marginally affected by function-blocking anti–&bgr;1 integrin antibodies, especially in the presence of PDGF-BB. In contrast, contraction mediated by &agr;V-negative mouse embryonic fibroblasts was completely blocked by anti–&bgr;1 integrin antibodies, even after stimulation with PDGF-BB. These results show a previously unrecognized in vivo function for the &agr;V&bgr;3 integrin, as a participant in the control of PIF during inflammatory reactions. Furthermore, our data demonstrate that PDGF-BB induces connective tissue cells to generate tensile forces via &agr;V&bgr;3 during such reactions.

High level of HSF1 associates with aggressive endometrial carcinoma and suggests potential for HSP90 inhibitors

Background:Recent identification of a specific role of HSF1 in cancer progression has led to new relevance of HSF1 as both a prognostic and a predictive marker. The role of HSF1 in endometrial cancer has so far been unexplored.Methods:A total of 823 lesions from endometrial carcinoma precursors, primary tumours and metastases were prospectively collected and explored for HSF1 protein expression in relation to established markers for aggressive disease and survival. Transcriptional alterations related to HSF1 protein level were investigated by microarray analysis for 224 freshly frozen samples in parallel.Results:High expression of HSF1 protein in endometrial carcinoma is significantly associated with aggressive disease and poor survival (all P-values ⩽0.02), also among ERα-positive patients presumed to have good prognosis. The HSF1-related gene signatures increase during disease progression and were also found to have prognostic value. Gene expression analyses identified HSP90 inhibition as a potential novel therapeutic approach for cases with high protein expression of HSF1.Conclusions:We demonstrate for the first time in endometrial cancer that high expression of HSF1 and measures for transcriptional activation of HSF1 associate with poor outcome and disease progression. The HSP90 inhibitors are suggested as new targeted therapeutics for patients with high HSF1 levels in tumour in particular.

Differential cytokine response in interstitial fluid in skin and serum during experimental inflammation in rats.

Tumour necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) are important mediators produced during inflammation. We hypothesized that the pro‐inflammatory cytokine response in the interstitial fluid (IF) is different from that in serum, and we aimed at quantifying the amount of TNF‐α and IL‐1β in the IF. By centrifugation of rat skin at < 424 g pure IF is extracted. Using ELISA such fluid was analysed for cytokines in back and/or paw skin of pentobarbital‐anaesthetized rats, after either induction of endotoxaemia or ischaemia–reperfusion (I/R) injury. During endotoxaemia, TNF‐α increased in the IF from 0 in control to 640 ± 100 pg ml−1 (mean ±s.e.m.) after 90 min, with the serum concentration being 5–10 times higher at all time points. The response pattern of IL‐1β after lipopolysaccharide (LPS) challenge differed greatly from that of TNF‐α with a large increase in IF from 390 ± 90 to 28 000 ± 1500 pg ml−1 after 210 min, and a significantly smaller increase in serum (600 ± 45 pg ml−1). During reperfusion of the hind paw after 2 h of ischaemia, there was a gradual increase of TNF‐α in both IF of the paw skin and serum after 3 min of reperfusion. Both declined after 20 min. The pattern for IL‐1β differed, increasing significantly less in serum (25 ± 15 pg ml−1 after 20 min of reperfusion) than in the IF (1100 ± 200 pg ml−1). Immunostaining of the inflamed tissues showed increased expression of the two cytokines in cells of both epidermis and dermis compared to controls. Subdermal injections of TNF‐α and IL‐1β at the same concentrations found in IF after LPS infusion affected interstitial fluid pressure significantly. Local TNF‐α production dominates after I/R injury, whereas in endotoxaemia systemic production predominates. For IL‐1β local production dominates in both conditions. Thus, there is a differential pattern of cytokine production and the current method allows the study of the role of cytokines in IF during different inflammatory reactions.

Effect of α-trinositol on interstitial fluid pressure, edema generation, and albumin extravasation after ischemia-reperfusion injury in rat hind limb

Reperfusion of ischemic tissue often leads to an acute inflammatory response, which acts directly to aggravate the injury in the reperfused zone, characterized by adhesion and subsequent infiltration of inflammatory cells that injure the tissue through the generation of oxygen-derived free radicals and release of various inflammatory mediators. The rapid edema formation associated with reperfusion injury is induced by increased microvascular permeability to plasma proteins and/or increased net filtration pressure across the microvascular wall, and the latter is at least in part induced by lowering of the interstitial fluid pressure (Pif). We investigated the anti-inflammatory effect of &agr;-trinositol (D-myo-inositol-1,2,6-trisphosphate) on edema formation, microvascular protein leakage, and Pif in rat hind limb after ischemia-reperfusion (I/R) injury. There was significant increase of both albumin extravasation from 0.02 ± 0.02 to 0.41 ± 0.21 mL g dry weight−1 (P < 0.05) and total tissue water from 1.08 ± 0.07 to 1.65 ± 0.55 mL g dry weight−1(P < 0.05) in the skin of paws undergoing I/R injury. Pif was significantly lowered from −0.51 ± 0.34 to −5.00 ± 1.53 mmHg (P < 0.05) concomitant with substantial edema formation. The edema formation, and lowering of Pif during I/R injury was significantly lowered and nearly totally abolished in the animals treated with &agr;-trinositol 30 min before reperfusion. We conclude that &agr;-trinositol limits the increased vascular permeability and edema formation by preventing the decrease in Pif as well as acting protective on the microvascular wall.

Effect of tumor necrosis factor-α, IL-1β, and IL-6 on interstitial fluid pressure in rat skin

Interstitial fluid pressure (Pif) decreases in several experimental models of acute inflammation, enhancing edema formation. The present study was designed to determine the effect of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β as well as lipopolysaccharides (LPS) on Pif in a model of gram-negative sepsis. Pif was measured in the paw skin of anesthetized rats (pentobarbital sodium, 50 mg/kg ip) using micropipettes (3-7 μm) and servo-controlled counterpressure technique. Test substances were injected intra-arterially (ia), intravenously (iv), or subdermally (sd). After intra-arterial or intravenous administration, the test substances were circulated for 1 min before circulatory arrest was induced with an intravenous injection of KCl while the rats were under pentobarbital anesthesia. Circulatory arrest was induced to avoid edema formation, which would raise interstitial fluid volume to cause a more positive Pif. Administration of 0.5 ml of LPS (5 mg/ml ia) lowered Pif significantly from control values of -0.2 ± 0.3 to -2.0 ± 0.3 mmHg ( P < 0.05) within 1 h. Corresponding values for TNF-α (500 ng/ml iv) were -0.4 ± 0.2 to -2.3 ± 0.1 mmHg ( P < 0.05). Administration of 5 μl (5 mg/ml sd) of LPS did not affect Pif significantly ( P > 0.05), but TNF-α, IL-1β, and IL-6 had a significant effect on Pif when given subdermally. IL-6 (50 ng/ml) caused a decrease in Pif from control values of -1.2 ± 0.3 to -2.8 ± 0.5 mmHg ( P < 0.05) within 1 h. The experiments demonstrate that LPS, TNF-α, IL-1β, and IL-6 induce lowering of Pif when given intravenously or intra-arterially, whereas only TNF-α, IL-1β, and IL-6 induce lowering of Pif when given subdermally. We therefore suggest that the lowering of Pif in this experimental model of sepsis is related to the release of and a local effect in skin of TNF-α, IL-1β, and IL-6.Interstitial fluid pressure (P(if)) decreases in several experimental models of acute inflammation, enhancing edema formation. The present study was designed to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta as well as lipopolysaccharides (LPS) on P(if) in a model of gram-negative sepsis. P(if) was measured in the paw skin of anesthetized rats (pentobarbital sodium, 50 mg/kg ip) using micropipettes (3-7 micrometer) and servo-controlled counterpressure technique. Test substances were injected intra-arterially (ia), intravenously (iv), or subdermally (sd). After intra-arterial or intravenous administration, the test substances were circulated for 1 min before circulatory arrest was induced with an intravenous injection of KCl while the rats were under pentobarbital anesthesia. Circulatory arrest was induced to avoid edema formation, which would raise interstitial fluid volume to cause a more positive P(if). Administration of 0.5 ml of LPS (5 mg/ml ia) lowered P(if) significantly from control values of -0.2 +/- 0.3 to -2.0 +/- 0.3 mmHg (P < 0.05) within 1 h. Corresponding values for TNF-alpha (500 ng/ml iv) were -0.4 +/- 0.2 to -2.3 +/- 0.1 mmHg (P < 0.05). Administration of 5 microliter (5 mg/ml sd) of LPS did not affect P(if) significantly (P > 0.05), but TNF-alpha, IL-1beta, and IL-6 had a significant effect on P(if) when given subdermally. IL-6 (50 ng/ml) caused a decrease in P(if) from control values of -1.2 +/- 0.3 to -2. 8 +/- 0.5 mmHg (P < 0.05) within 1 h. The experiments demonstrate that LPS, TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given intravenously or intra-arterially, whereas only TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given subdermally. We therefore suggest that the lowering of P(if) in this experimental model of sepsis is related to the release of and a local effect in skin of TNF-alpha, IL-1beta, and IL-6.

Intimal thickness of the coronary arteries in low-birthweight infants.

Background: Intimal thickening is considered to be an early manifestation of developing atherosclerosis in healthy young adults and children. Low birthweight correlates with increased incidence of cardiovascular diseases. Aim: To test the hypothesis that low birthweight is associated with relatively thickened intima at birth. Methods: The coronary arteries of 175 children were screened from serial cross‐sections for maximal intimal thickening and measured morphometrically. The area of intima and media and the length of internal elastic lamina were measured. The intimal to medial area ratio and calculated thicknesses of intima were used in statistical comparisons. Only children who died within 30 d after birth (n=111) were included. Results: There was a significant positive correlation between intimal thickness and birthweight in low‐birthweight children (p <0.006). Neither the relative thickness of the intima nor the ratio of intimal to medial area increased with increasing growth restriction. The sum of the thicknesses of arterial media and intima had a significant positive correlation with birthweight in these infants.

α-Trinositol prevents increased negativity of interstitial fluid pressure in rat skin and trachea induced by dextran anaphylaxis

The new anti-inflammatory agent alpha-trinositol (D-myo-inositol-1,2,6-trisphosphate), is suggested to act on the cellular adhesion receptor towards extracellular matrix components, the beta1-integrins, and may therefore represent a novel principle for therapy of the phenomena associated with acute inflammation. Increased negativity of interstitial fluid pressure (p(if)) is a major driving force for the rapid edema formation in trachea and skin associated with dextran anaphylaxis in the rat. We therefore used this experimental model to study the effect of alpha-trinositol in skin and trachea of pentobarbital anesthetized rats. p(if) was measured with sharpened glass capillaries (3-7 microm) connected to a servocontrolled counterpressure system. alpha-Trinositol (10 mg) was given before or after dextran. Circulatory arrest was induced 2 min after i.v. dextran to limit the increased capillary fluid filtration associated with the anaphylactic reaction. This increased filtration will otherwise raise interstitial volume and thereby p(if) and cause an underestimation of a potential increased negativity of p(if). In the trachea, p(if) was 0.0 +/- 1.0 mmHg (S.D.) and -1.4 +/- 0.5 mmHg in controls given saline vehicle and alpha-trinositol (P > 0.05), respectively, and fell to -8.5 +/- 2.7 mmHg after dextran (P < 0.01). alpha-Trinositol given 2 min prior to or after dextran resulted in p(if) of -1.7 +/- 1.2 mmHg (P > 0.05 versus control, P < 0.01 versus dextran) and -4.7 +/- 3.0 mmHg (P < 0.01 versus control and dextran), respectively. In skin, i.v. dextran caused p(if) to fall from -0.6 +/- 0.5 to -4.6 +/- 1.9 mmHg (P < 0.001). When alpha-trinositol was given prior to dextran the corresponding figures were -0.4 +/- 0.8 and -0.9 +/- 1.1 mmHg, respectively (P > 0.05). Subdermal administration of alpha-trinositol after i.v. dextran and circulatory arrest normalized p(if) in concentration of 100, 10 and partly at 1 mg/ml. Thus, alpha-trinositol prevented the increased negativity of p(if) induced by dextran anaphylaxis when administered prior to as well as after dextran showing that the alpha-trinositol also could influence an already started inflammatory reaction.