Rolf K. Reed

Vasostatins, Comprising the N-terminal Domain of Chromogranin A, Suppress Tension in Isolated Human Blood Vessel Segments

Chromogranin A (CGA) belongs to a family of highly acidic proteins which are co‐stored and co‐released with the catecholamines from the mammalian adrenal gland and occur in nmolar concentrations in the human circulation. A vascular function for the adrenomedullary released and circulating CGA has yet to be established. The present study reports on the novel vasoinhibitory effect of the N‐terminal domain of the adrenomedullary CGA in isolated segments of the human internal thoracic artery (ITA) and saphenous vein (SV). The collective term vasostatin(s) refers to N‐terminal fragments (CGA1–76 and CGA1–113) of apparent molecular weights 7 to 22 kD, to indicate their vascular inhibitory effects. The sustained contractions evoked by the potent vasoconstrictor peptide, endothelin‐1 (ET‐1) were suppressed when ITA and SV segments were preincubated for 15 min with vasostatins (72 nM). The vasoinhibitory effects were not dependent on an intact endothelium and suppression of the response to 35 nM ET‐1 was ∼77% and ∼40% in endothelium‐denuded ITA and SV segments, respectively. In endothelium‐denuded SV segments the vasostatins suppressed the maximal sustained tension response but not the potency for ET‐1, indicating that the vasostatin effect did not involve interference with ET‐1 binding to its vascular receptor. Preincubation of endothelium‐denuded SV segments with nifedipine (1 μM) inhibited the sustained response to ET‐1 ≥10 nM by 50%. By the same protocol a 20 to 40% suppression of the contraction evoked by ET‐1 ≥10 nM was obtained in segments preincubated with vasostatins (72 nM), suggesting that inhibition of Ca2 channel opening might indirectly contribute to the effect of vasostatins on the sustained response to ET‐1. The mechanisms for these inhibitory effects of vasostatins on the sustained ET‐1 contractions in human blood vessels remain to be elucidated.

Interstitial fluid pressure in rats measured with a modified wick technique.

Abstract Subcutaneous interstitial fluid pressure (Pi) was measured in anesthetized rats with the “wick-in-needle” technique: A thin hypodermic needle was provided with a 2- to 4-mm-long sidehole and filled with multifilamentous nylon thread. A polyethylene tube, connecting the needle to a pressure transducer, could be compressed by a screw clamp, displacing a volume of about 0.2 μl. Compression caused a marked rise in pressure, but return to control level was obtained within 1 to 5 min. Decompression caused opposite pressure changes, confirming satisfactory fluid communication between needle and tissue fluid. Control Pi (measured with saline-filled needle) ranged from −2.5 to +1.0 mm Hg, with mean values of −0.65 mm Hg (SD 0.82, n = 44) on the back and −1.2 mm Hg (SD 0.8, n = 11) on the hindlimb. Similar values were obtained with the needle filled with serum or 1% hyaluronic acid. Severe reduction of femoral arterial pressure reduced hindlimb Pi gradually to −3 to −4 mm Hg in the course of 1 to 2 hr. Pi was also reduced after 3–7 days of dehydration (−2 to −6 mm Hg) and rose rapidly with rehydration. Edema and a rise of hindlimb Pi to positive values were observed when femoral venous pressure was increased from the control level of 3 to 12 mm Hg or more for a period of 24 hr.

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.

New and active role of the interstitium in control of interstitial fluid pressure: potential therapeutic consequences.

Here we present recent data indicating that the present view of the interstitium as a passive fluid reservoir has to be revised. The connective tissue cells and extracellular matrix have a role in the control of Pif and a fundamental role in the rapid development of edema in burns and in the initial swelling in inflammation by generating a lowering of interstitial fluid pressure. In this process, the β1‐integrin system seems to provide a common pathway by which the cells can lower as well as raise Pif. Inflammatory swelling can be reversed by endo‐ and exogenous substances, thereby suggesting that the connective tissue can serve as a novel target for pharmacological intervention. Furthermore, the new knowledge in interstitial physiology on means to reduce interstitial fluid pressure may be of importance for drug delivery into solid tumors, where a high Pif limits the uptake of therapeutic agents.

Micropuncture measurement of interstitial fluid pressure in rat subcutis and skeletal muscle: Comparison to wick-in-needle technique

Abstract Interstitial fluid pressure (IFP) in subcutis and skeletal muscle of rats has been measured by sharpened glass capillaries (diameter, 1–3 μm) connected to a servocontrolled counterpressure system (Wiederhielm). Measurements in subcutis were made by a puncture through intact skin, while a minute skin incision was made for measurements in skeletal muscle. Average IFP was −1.09 mm Hg (SD 0.58, n = 120) in subcutis and −0.12 mm Hg (SD 0.65, n = 129) in skeletal muscle. Simultaneous measurements with micropipets and with “wick-in-needle” gave practically identical mean pressures, and showed good correlation under varying degrees of hydration and dehydration in subcutis ( r = 0.92) and in skeletal muscle ( r = 0.91). Both methods showed unaltered pressure in both tissues during the first hour after killing the rat. In vitro studies showed that the micropipet recorded about 6% of the colloid osmotic pressure of a 1–2% hyaluronic acid gel. Hydrostatic pressures in the range of −10 to +10 mm Hg imposed on a 1% hyaluronic acid gel were exactly reflected by the micropipet, indicating that the feasibility of micropuncture measurements of tissue pressure does not exclude a continuous gel phase in the interstitium. While the practical use of micropuncture measurements may be limited by the requirement of anesthesia and immobilization of the tissue, it should serve as a useful reference for pressures measured by more versatile methods.

Lowering of tumor interstitial fluid pressure specifically augments efficacy of chemotherapy

Chemotherapy of solid tumors is presently largely ineffective at dosage levels that are compatible with survival of the patient. Here, it is argued that a condition of raised interstitial fluid pressure (IFP) that can be observed in many tumors is a major factor in preventing optimal access of systemically administered chemotherapeutic agents. Using prostaglandin E1‐methyl ester (PGE1), which is known transiently to reduce IFP, it was shown that 5‐fluorouracil (5‐FU) caused significant growth inhibition on two experimental tumors in rats but only after administration of PGE1. Furthermore, timing experiments showed that only in the period in which IFP is reduced did 5‐FU have an antitumor effect. These experiments uniquely demonstrate a clear and, according to the starting hypothesis, logical, synergistic effect of PGE1 and 5‐FU that offers hope for better treatment of many tumors in which raised IFP is likely to be inhibiting optimal results with water‐soluble cancer chemotherapeutic agents.

Transcapillary exchange: Role and importance of the interstitial fluid pressure and the extracellular matrix

This review will summarize current knowledge on the role of the extracellular matrix (ECM) in general and on the interstitial fluid pressure (P(if)) in particular with regard to their importance in transcapillary exchange. The fluid volume in the interstitial space is normally regulated within narrow limits by automatic re-adjustment of the interstitial hydrostatic and colloid osmotic pressures in response to perturbations in capillary filtration and by the lymphatics. Contrary to this commonly accepted view, P(if) can become an active force and create a fluid flux across the capillaries in several inflammatory reactions and trauma situations rather than limit the changes occurring. The molecular mechanisms involved in the lowering of P(if) include the release of cellular tension exerted on the collagen and microfibril networks in the connective tissue via the collagen-binding beta(1)-integrins, thereby allowing the glycosaminoglycan ground substance, which is normally underhydrated, to expand and take up fluid. Several growth factors and cytokines, including the platelet-derived growth factor BB, are able to reverse a lowering of P(if) and restore the normal compaction of the ECM. The magnitude of the lowering of P(if) varies with the inflammatory response. In several inflammatory reactions, a lowering of P(if) to -5 to -10 mmHg is seen, which will increase capillary filtration by 10-20 times since the normal capillary filtration pressure is usually 0.5-1 mmHg (skin and skeletal muscle). Unless this lowering of P(if) is taken into account, the enhanced solute flux resulting from an inflammatory response will be ascribed to an increased capillary permeability.

Catabolism of hyaluronan in rabbit skin takes place locally, in lymph nodes and liver

The catabolism of hyaluronan has been studied by injecting hyaluronan, labelled with 125I‐tyramine cellobiose (125I‐TC), subcutaneously into the hindpaw of rabbits. Following endocytosis, 125I‐TC remains in the cells at the site of uptake, allowing localization of the site of catabolism. At 6 h after subcutaneous injection, 65% of the injected radioactivity was recovered. The skin at the injection site contained 47%, the popliteal gland at the side of injection 10%, and the liver 8% of the injected dose. At 48 h the three organs contained 40% of the injected dose with 17% in the skin, 10% in the lymph node and 13% in the liver. The decline in recovery could be accounted for by urinary excretion of the tracer, implying that some tracer had been released from the cells after endocytosis. Chromatography revealed that over 85% of 125I‐TC‐hyaluronan in the lymph nodes and liver was of low molecular mass throughout the experiment. In skin, 4% of the injected tracer was recovered with low molecular mass at 6 h, increasing to 12% of injected dose at 24 and 48 h. Thus, a minimum of 12% of the injected tracer was catabolized per 24 h at the skin injection site. If cells in skin are responsible for the subsequent release of tracer, as seen from the decrease in recovery of the injected dose, another 10‐15% of the tracer could have been catabolized locally in the skin per day. The major part of the hyaluronan injected in the skin was, however, catabolized by lymphatic removal and subsequent degradation in local lymph nodes and liver.

Targeting the NG2/CSPG4 Proteoglycan Retards Tumour Growth and Angiogenesis in Preclinical Models of GBM and Melanoma

Aberrant expression of the progenitor marker Neuron-glia 2 (NG2/CSPG4) or melanoma proteoglycan on cancer cells and angiogenic vasculature is associated with an aggressive disease course in several malignancies including glioblastoma multiforme (GBM) and melanoma. Thus, we investigated the mechanism of NG2 mediated malignant progression and its potential as a therapeutic target in clinically relevant GBM and melanoma animal models. Xenografting NG2 overexpressing GBM cell lines resulted in increased growth rate, angiogenesis and vascular permeability compared to control, NG2 negative tumours. The effect of abrogating NG2 function was investigated after intracerebral delivery of lentivirally encoded shRNAs targeting NG2 in patient GBM xenografts as well as in established subcutaneous A375 melanoma tumours. NG2 knockdown reduced melanoma proliferation and increased apoptosis and necrosis. Targeting NG2 in two heterogeneous GBM xenografts significantly reduced tumour growth and oedema levels, angiogenesis and normalised vascular function. Vascular normalisation resulted in increased tumour invasion and decreased apoptosis and necrosis. We conclude that NG2 promotes tumour progression by multiple mechanisms and represents an amenable target for cancer molecular therapy.

Catabolism of hyaluronan in the knee joint of the rabbit.

Catabolism of hyaluronan was studied by injecting hyaluronan labelled with [125I]-tyramine cellobiose ([125I]-TC) into knee joints of rabbits. After endocytosis [125I]-TC remains intracellularly allowing localization of the site of catabolism. At 6 hours after injection 63% could be recovered in and around the joint, while at 48 hours 32% remained locally. Chromatography showed that 12% of the injected tracer was degraded in joint tissues at 6 hours, increasing to 33% at 24 hours. There was no apparent degradation within the joint fluid. No tracer was found in the regional lymph glands, but 16% of the injected tracer was detected in the liver at 24 hours. This investigation demonstrates that hyaluronan in the joint can be degraded both locally and in the liver.