Kristian Pietras

High interstitial fluid pressure — an obstacle in cancer therapy

Many solid tumours show an increased interstitial fluid pressure (IFP), which forms a barrier to transcapillary transport. This barrier is an obstacle in tumour treatment, as it results in inefficient uptake of therapeutic agents. There are a number of factors that contribute to increased IFP in the tumour, such as vessel abnormalities, fibrosis and contraction of the interstitial matrix. Lowering the tumour IFP with specific signal-transduction antagonists might be a useful approach to improving anticancer drug efficacy.

Protein detection using proximity-dependent DNA ligation assays

The advent of in vitro DNA amplification has enabled rapid acquisition of genomic information. We present here an analogous technique for protein detection, in which the coordinated and proximal binding of a target protein by two DNA aptamers promotes ligation of oligonucleotides linked to each aptamer affinity probe . The ligation of two such proximity probes gives rise to an amplifiable DNA sequence that reflects the identity and amount of the target protein. This proximity ligation assay detects zeptomole (40 × 10−21 mol) amounts of the cytokine platelet-derived growth factor (PDGF) without washes or separations, and the mechanism can be generalized to other forms of protein analysis.

PDGF-D is a specific, protease-activated ligand for the PDGF beta-receptor

The term platelet-derived growth factor (PDGF) refers to a family of disulphide-bonded dimeric isoforms that are important for growth, survival and function in several types of connective tissue cell. So far, three different PDGF chains have been identified — the classical PDGF-A and PDGF-B and the recently identified PDGF-C. PDGF isoforms (PDGF-AA, AB, BB and CC) exert their cellular effects by differential binding to two receptor tyrosine kinases. The PDGF α-receptor (PDGFR-α) binds to all three PDGF chains, whereas the β-receptor (PDGFR-β) binds only to PDGF-B. Gene-targeting studies using mice have shown that the genes for PDGF-A and PDGF-B, as well as the two PDGFR genes, are essential for normal development. Furthermore, overexpression of PDGFs is linked to different pathological conditions, including malignancies, atherosclerosis and fibroproliferative diseases. Here we have identify and characterize a fourth member of the PDGF family, PDGF-D. PDGF-D has a two-domain structure similar to PDGF-C and is secreted as a disulphide-linked homodimer, PDGF-DD. Upon limited proteolysis, PDGF-DD is activated and becomes a specific agonistic ligand for PDGFR-β . PDGF-DD is the first known PDGFR-β-specific ligand, and its unique receptor specificity indicates that it may be important for development and pathophysiology in several organs.

A Multitargeted, Metronomic, and Maximum-Tolerated Dose “Chemo-Switch” Regimen is Antiangiogenic, Producing Objective Responses and Survival Benefit in a Mouse Model of Cancer

PURPOSEnA transgenic mouse model has revealed parameters of the angiogenic switch during multistep tumorigenesis of pancreatic islets, and demonstrated efficacy of antiangiogenic therapies. Pericytes have been revealed as functionally important for tumor neovasculature, using kinase inhibitors targeting their platelet-derived growth factor receptors (PDGFRs). Additionally, vascular endothelial growth factor receptor (VEGFR) inhibitors and metronomic chemotherapy show modest benefit against early- but not late-stage disease.nnnMATERIALS AND METHODSnSeeking to improve efficacy against otherwise intractable end-stage pancreatic islet tumors, two receptor tyrosine kinase inhibitors, imatinib and SU11248, were used to disrupt PDGFR-mediated pericyte support of tumor endothelial cells in concert with maximum-tolerated dose (MTD) or metronomic chemotherapy and/or VEGFR inhibition.nnnRESULTSnImatinib, despite equivocal efficacy as monotherapy, reduced pericyte coverage of tumor vessels and enhanced efficacy in combination with metronomic chemotherapy or VEGFR inhibition. A regimen involving all three was even better. MTD using cyclophosphamide caused transitory regression, but then rapid regrowth, in contrast to metronomic cyclophosphamide plus imatinib, which produced stable disease. The MTD regimen elicited apoptosis of tumor cells but not endothelial cells, whereas the other regimens increased endothelial cell apoptosis concordant with efficacy. A chemo-switch protocol, involving sequential MTD and then metronomic chemotherapy, overlaid with multitargeted inhibition of PDGFR and VEGFR, gave complete responses and unprecedented survival advantage in this model.nnnCONCLUSIONnThis study demonstrates a potentially tractable clinical strategy in a stringent preclinical model, wherein standard-of-care chemotherapy is followed by a novel maintenance regimen: PDFGR is targeted to disrupt pericyte support, while metronomic chemotherapy and/or VEGFR inhibitors target consequently sensitized endothelial cells, collectively destabilizing pre-existing tumor vasculature and inhibiting ongoing angiogenesis.

PDGF receptors as cancer drug targets.

The benefits of combining PDGF antagonists and VEGF antagonists in a mouse model of pancreatic islet cancer are shown in a report by Bergers et al.: Bergers, G., Song, S., Meyer-Morse, N., Bergsland, E., and Hanahan, D. (2003). Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. 111, 1287–1295.

Vascular endothelial growth factor B controls endothelial fatty acid uptake

The vascular endothelial growth factors (VEGFs) are major angiogenic regulators and are involved in several aspects of endothelial cell physiology. However, the detailed role of VEGF-B in blood vessel function has remained unclear. Here we show that VEGF-B has an unexpected role in endothelial targeting of lipids to peripheral tissues. Dietary lipids present in circulation have to be transported through the vascular endothelium to be metabolized by tissue cells, a mechanism that is poorly understood. Bioinformatic analysis showed that Vegfb was tightly co-expressed with nuclear-encoded mitochondrial genes across a large variety of physiological conditions in mice, pointing to a role for VEGF-B in metabolism. VEGF-B specifically controlled endothelial uptake of fatty acids via transcriptional regulation of vascular fatty acid transport proteins. As a consequence, Vegfb-/- mice showed less uptake and accumulation of lipids in muscle, heart and brown adipose tissue, and instead shunted lipids to white adipose tissue. This regulation was mediated by VEGF receptor 1 and neuropilin 1 expressed by the endothelium. The co-expression of VEGF-B and mitochondrial proteins introduces a novel regulatory mechanism, whereby endothelial lipid uptake and mitochondrial lipid use are tightly coordinated. The involvement of VEGF-B in lipid uptake may open up the possibility for novel strategies to modulate pathological lipid accumulation in diabetes, obesity and cardiovascular diseases.

Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting.

Background Important support functions, including promotion of tumor growth, angiogenesis, and invasion, have been attributed to the different cell types populating the tumor stroma, i.e., endothelial cells, cancer-associated fibroblasts, pericytes, and infiltrating inflammatory cells. Fibroblasts have long been recognized inside carcinomas and are increasingly implicated as functional participants. The stroma is prominent in cervical carcinoma, and distinguishable from nonmalignant tissue, suggestive of altered (tumor-promoting) functions. We postulated that pharmacological targeting of putative stromal support functions, in particular those of cancer-associated fibroblasts, could have therapeutic utility, and sought to assess the possibility in a pre-clinical setting. Methods and Findings We used a genetically engineered mouse model of cervical carcinogenesis to investigate platelet-derived growth factor (PDGF) receptor signaling in cancer-associated fibroblasts and pericytes. Pharmacological blockade of PDGF receptor signaling with the clinically approved kinase inhibitor imatinib slowed progression of premalignant cervical lesions in this model, and impaired the growth of preexisting invasive carcinomas. Inhibition of stromal PDGF receptors reduced proliferation and angiogenesis in cervical lesions through a mechanism involving suppression of expression of the angiogenic factor fibroblast growth factor 2 (FGF-2) and the epithelial cell growth factor FGF-7 by cancer-associated fibroblasts. Treatment with neutralizing antibodies to the PDGF receptors recapitulated these effects. A ligand trap for the FGFs impaired the angiogenic phenotype similarly to imatinib. Thus PDGF ligands expressed by cancerous epithelia evidently stimulate PDGFR-expressing stroma to up-regulate FGFs, promoting angiogenesis and epithelial proliferation, elements of a multicellular signaling network that elicits functional capabilities in the tumor microenvironment. Conclusions This study illustrates the therapeutic benefits in a mouse model of human cervical cancer of mechanism-based targeting of the stroma, in particular cancer-associated fibroblasts. Drugs aimed at stromal fibroblast signals and effector functions may prove complementary to conventional treatments targeting the overt cancer cells for a range of solid tumors, possibly including cervical carcinoma, the second most common lethal malignancy in women worldwide, for which management remains poor.

Angiogenesis stimulated by PDGF-CC, a novel member in the PDGF family, involves activation of PDGFR-αα and -αβ receptors

A newly discovered PDGF isoform, PDGF‐CC, is expressed in actively angiogenic tissues such as placenta, some embryonic tissues, and tumors. We test the possibility that PDGF‐CC promotes angiogenesis in vivo. The core domain (mature form) of human PDGF‐CC is sufficiently potent to stimulate neovascularization in the mouse cornea. The corneal angiogenic response induced by PDGF‐CC is robust although the area of neovascularization is smaller than those of FGF‐2‐and VEGF‐stimulated angiogenesis. Similarly, PDGF‐BB and PDGF‐AB induce angiogenic responses virtually indistinguishable from PDGF‐CCstimulated vessels. In contrast, PDGF‐AA displays only a weak angiogenic response in the mouse cornea. Although there was no significant difference in incorporation of mural cells to the newly formed blood vessels induced by PDGF‐BB and ‐CC, the percentage of mural cell positive vessels induced by PDGF‐AA was greater than those induced by FGF‐2, PDGF‐BB, and PDGF‐CC. In the developing chick embryo, PDGF‐CC induced branch sprouts from established blood vessels. In PDGF receptor‐transfected endothelial cells, PDGF‐CC activated the PDGF receptor alpha subunit (PDGFR‐a). PDGF‐CC, but not PDGF‐AA, was able to activate PDGFR‐p receptor in endothelial cells that coexpress both α and β forms of receptors. Thus, the PDGF‐CC‐mediated angiogenic response is most likely transduced by PDGF‐aa and ‐ap receptors. These data demonstrate that the PDGF family is a complex and important group of proangiogenic factors.—Cao, R., Bråkenhielm, E., Li, X., Pietras, K., Widenfalk, J, Östman, A., Eriksson, U., Cao, Y. Angiogenesis stimulated by PDGF‐CC, a novel member in the PDGF family, involves activation of PDGFR‐aa and ‐ap receptors. FASEB J. 16, 1575–1583 (2002)

Platelet-Derived Growth Factor D Induces Cardiac Fibrosis and Proliferation of Vascular Smooth Muscle Cells in Heart-Specific Transgenic Mice

Platelet-derived growth factor (PDGF)-D is a member of the PDGF/vascular endothelial growth factor family that activates PDGF receptor β (PDGFR-β). We show that PDGF-D is highly expressed in the myocardium throughout development and adulthood, as well as by arterial vascular smooth muscle cells (vSMCs). To obtain further knowledge regarding the in vivo response to PDGF-D, we generated transgenic mice overexpressing the active core domain of PDGF-D in the heart. Transgenic PDGF-D stimulates proliferation of cardiac interstitial fibroblasts and arterial vSMCs. This results in cardiac fibrosis followed by dilated cardiomyopathy and subsequent cardiac failure. Transgenic mice also display vascular remodeling, including dilation of vessels, increased density of SMC-coated vessels, and proliferation of vSMCs, leading to a thickening of tunica media. The thickening of arterial walls is a unique feature of PDGF-D, because this is not seen when PDGF-C is overexpressed in the heart. These results show that PDGF-D, via PDGFR-β signaling, is a potent modulator of both vascular and connective tissue growth and may provide both paracrine and autocrine stimulation of PDGFR-β. Our data raise the possibility that this growth factor may be involved in cardiac fibrosis and atherosclerosis.

Increased Vascular Delivery and Efficacy of Chemotherapy after Inhibition of Platelet-Derived Growth Factor-B

Inhibition of platelet-derived growth factor-B (PDGF-B) has multiple effects on tumors, including loss of pericytes, regression of some vessels, normalization of other vessels, and reduction of interstitial pressure. PDGF-B inhibition also increases the efficacy of cancer therapeutics, but the role on tumor vessel efficiency and drug delivery is unclear. We sought to determine whether inhibition of PDGF-B signaling can increase delivery and efficacy of cyclophosphamide in Lewis lung carcinomas or RIP-Tag2 tumors. PDGF-B blockade in Lewis lung carcinoma tumors by the DNA aptamer AX102 for 14 days increased the number of perfused tumor vessels marked by lectin in the bloodstream by 50%. AX102 also increased the width of sleeves of viable tumor cells around blood vessels by 66%, increased tumor cell proliferation by 90%, and increased intratumoral delivery of Hoechst 33342 by 78%. A low dose of cyclophosphamide (20 mg/kg) reduced tumor cell proliferation by 31% when combined with AX102 but not when given alone. Synergy of cyclophosphamide and AX102 on tumor cell proliferation also was found in RIP-Tag2 tumors. Similarly, the PDGF receptor signaling inhibitor imatinib increased delivery of cyclophosphamide and reduced tumor burden in RIP-Tag2 mice, without evidence of tumor cell sensitization to chemotherapy. Together, these findings indicate that inhibition of PDGF-B signaling promotes the delivery and efficacy of chemotherapeutic agents by increasing the efficiency of tumor blood vessels.