VEGF-D on Michael’s Domain

An impossible overlap-extension PCR

Sat, 21 Jun 2025 00:00:00 +0000

A PhD student of mine asked me about plasmid maps for several DNA constructs that I had created some 20 years ago. Since SnapGene did not exist at the time, I had used the now obsolete GeneConstructionKit2. I performed many clonings at the time, but I did not continue generating maps for all of them. The GCK2 format does not allow for easy annotation. You needed a separate program for comprehensively annotating the plasmids, and this data was saved in a separate file, the so-called “illustration” file: what could possibly go wrong? Yesterday, I ended up digging out my old lab notebooks and retracing about 10 old clonings in SnapGene. However, I was unable to simulate one of the assemblies because SnapGene was too conservative in disallowing “bad” PCR primers to function. I had performed overlap-extension PCR to introduce a mutation into the mouse VEGF-D cDNA. The homologous mutation had been introduced into human VEGF-D before, and I therefore had the primers for the human sequences. Mouse and human VEGF-D are very similar. The primers designed to amplify the human PCR were not perfect when using mouse cDNA as a template, but none of the differences would result in amino acid changes. So I attempted the PCR with a primer that had a mismatch in the third nucleotide from the 3’-end. The PCR was successful, but even when I lowered the hybridisation parameters to the least stringent settings, SnapGene would not anneal this primer to my template. To simulate cloning in SnapGene and generate a map, I needed to introduce a mutation into my primer and then reverse the mutation after the overlap extension PCR. I guess I need to file a bug report (or would this be a feature request?). It seems appropriate that the program should be able to allow annealing of primers that do anneal in reality…

Our lab won't go bancrupt in 2025!

Fri, 29 Nov 2024 00:00:00 +0000

Our lab has been so unlucky with its recent grant applications that I was already seriously considering alternative career paths. Like becoming a tram driver. Helsinki and its surrounding cities are expanding their tram networks, and good drivers are rare. However, the Cancer Foundation Finland has now ended our unlucky streak! Although it is only a modest amount, I am hopeful for the future. Not all research is equally expensive: some methods need more money than others, and ours are at the budget end of that spectrum. What project got funded? Well, it’s not our novel protein expression system, but a project to answer a question that has been bugging many vascular biology researchers since 2001, when the Achen/Stacker lab published a paper showing that mouse and human VEGF-D do NOT share the same receptors, but that mouse VEGF-D does not bind mouse VEGFR-2 ( Baldwin et al., 2001 ). We showed in 2011 and 2019 , that the proteolytic activation of VEGF-D by cathepsin D leads to a loss of all VEGFR-3 binding in human VEGF-D. If this was true for mouse VEGF-D as well, it would be a growth factor without a receptor! This is not impossible, but it would be exceptional. It is important to get these molecular details right because we are testing our future cancer drugs always in mice. If there is a big difference in the angiogenic signaling between mice and men, this could render much mouse data very difficult to interpret and perhaps even explain why drugs that work well in mice fail in humans. Notably, among all drugs, oncology drugs have the worst success rates in clinical trials. There is reasonable evidence to believe that VEGF-D is the “bad boy” in the VEGF family, i.e., the one that is responsible for cancers developing resistance against antiangiogenic cancer therapies, that are based on blocking VEGF-A. In any case, a big shout-out to the Cancer Foundation Finland! Read here about the award.

KLK3: tumorigenic or not?

Wed, 22 Dec 2021 00:00:00 +0000

We have just published our latest review about the role of KLK3 as an activator of VEGF-C and VEGF-D in prostate cancer . Prostate cancer is one of the most common cancers in males. It is not a question of whether you will get it but only when. Once you reach your 80s, the likelihood of you having prostate cancer is bigger than not having it. In a 2013 autopsy study of Japanese males , who died of other causes, 59% of those older than 80 had prostate cancer. It is likely that many of these cases were indolent and would never have caused any problems. Only a few of them might have become symptomatic had these men lived longer. So, there is a significant interest in distinguishing those cancers that are going to cause problems. Many prognostic markers have been proposed to do exactly that: to predict which cancers would become problematic.From the vascular biology point of view, angiogenesis and lymphangiogenesis are two hallmarks of cancers that have been previously proposed to have prognostic value. When we stumbled upon the fact that prostate-specific antigen (PSA, also known as KLK3) is able to activate VEGF-C and VEGF-D , we thought that this might have significance for prostate cancer. Meanwhile, further research has clarified some questions, and it really seems to be that both VEGF-C and VEGF-D are involved in cancer progression. it is not clear yet which proteases are responsible for the activation of VEGF-C and VEGF-D in real human cancers. KLK3- or Cathepsin D (CTSD)-activated VEGF-D might be a possible cause of the resistance of tumors to bevacizumab (Avastin) treatment. The consequences of VEGF-C activation, on the other hand, are more difficult to predict because activated VEGF-C does simultaneously both good and bad: On the one hand, it facilitates metastasis. On the other hand, it enables an enhanced immune response against the tumour. Interesting research lies ahead. Read more in our review: https://doi.org/10.3390/ijms222413545

KLK3/PSA and cathepsin D activate VEGF-C and VEGF-D

Sat, 18 May 2019 00:00:00 +0000

Prostate-specific antigen (PSA) is well known - at least among older males - as a prostate cancer marker, but few people know its physiological function: Sperm cells are trapped in fresh ejaculate, which has a jelly-like consistence. In order to release the sperm cells, the ejaculate needs to be liquefied and precisely this liquefaction is the task of PSA.Also surprising for many people is the fact, that scientists still do not know why high PSA levels are associated with prostate cancer. In our latest research published yesterday in eLIFE , we have made a big step ahead in understanding the role of PSA in both reproductive and cancer biology.It appears that PSA (aka as kallikrein-related peptidase 3 - KLK3) and another enzyme called cathepsin D can activate two growth factors which have been implicated in cancer progression: VEGF-C and VEGF-D . These growth factors do likely contribute to tumor angiogenesis and tumor lymphangiogenesis. By inducing angiogenesis - the growth of blood vessels - the tumor ensures its own supply with nutrients and oxygen. Such blood supply is necessary for a tumor to grow beyond the size of a few millimeters. Likewise, tumor lymphangiogenesis happens when the tumor induces the growth of lymphatic vessels and it is tightly linked to the lymphatic spread (metastasis) of the tumor.Both VEGF-C and VEGF-D are produced as inactive precursors (pro-VEGF-C, pro-VEGF-D) and need to be activated in order to induce the growth of blood or lymphatic vessels. With ADAMTS3 , we have identified the enzyme that activates VEGF-C during embryonic development - which also requires vessel growth - in 2014 ( Jeltsch et al. ). However, it remained unclear whether the same enzyme is responsible also for pathological vessel growth. Now it seems likely that patholigical vessel growth uses different enzymes and PSA and cathepsin D have become prime suspects. Our next experiments will test whether we can slow down or halt cancer growth by blocking these enzymes.

Self Archiving and Open Access

Sun, 07 Jul 2013 00:00:00 +0000

I recently wrote a review article for the journal Lymphologie in Forschung ind Praxis . Its title was “Die lymphangiogenen Wachstumsfaktoren VEGF-C und VEGF-D” and it was the first paper I wrote in my mother tongue, German. This journal’s impact factor has been consistently below 1, which is not uncommon for non-English journals. However, in the big European countries like Germany, France and Italy, there are still many doctors who are not comfortable reading English. I though I’d help them out catching up on the latest in lymphatic research. Opening up access to science and visibility of science is all good, so I thought.Because my boss argued that it was a waste of time (I hope to prove him wrong - help me out here DLG !), I minimized the effort by engaging another knowledgeable German researcher at the University of Helsinki. Luckily I had already a draft when I was asked to write the review, even though I started to write it about two years ago and it was targeted for my website. When the article was published I received two physical reprints. When I tried linking to the online version of the article, I had to realize that it was behind a paywall . Because most publishers nowadays support self archiving , I asked the publisher about their policy. I presume that the publisher did not have any policy in place concerning self archiving, because they said they would agree to it, but I would have to get the green light from the board of directors of the DLG .I really hope to get this permission because this is the only way I can fulfill reprint request without hassle (yes I could copy the pages and send them by post (but aren’t we living in the 21st century?). If I won’t get the permission, one legal and easy way to distribute this article would be to put a pre-print version (i.e. the manuscript that I wrote) online. Luckily the copyrights of the publisher cover only the published version and not the pre-print versions. Others have done it this way (and they attached a list of the changes, that were made to make the pre-print version identical to the published version). This is a suboptimal solution, but maximizing accessibility and visibility. My University has a loose requirement to publish only in Open Access journals. However, exceptions to this policy of the University of Helsinki concerning Open Access are made on a regular basis, but they will get more and more difficult as the EU tightens their funding policy including the requirements for Open Access to research results . According to the EU’s interpretation, a journal could be considered Open Access if it allows for self archiving of the published article, self archiving being the second, “green” route to Open Access. The Academy of Finland (my main funding source) has a similar interpretation: “We further recommend that Academy-funded researchers publish their articles in open-access scientific journals, if there are online journals in the field in question that are at least of the same high quality as traditional subscription journals. The articles can also be saved in open-access electronic archives.” For employees of Helsinki University, self archiving is mandatory since 2010 .That makes sense to me: As a scientist I work with tax payers’ money; therefore all tax payers should have access to the results of my work. Even though I worked for this review only in my spare time, technically the requirements still apply as I used a computer, that was paid with tax payers’ money… Stay tuned and if you need the article now (and don’t want to wait for the DLG to decide), please e-mail me!

Unlocking the drains

Mon, 01 Aug 2005 00:00:00 +0000

Nice article by Phyllida Brown in Nature describing the discovery of the VEGF-C/VEGFR-3 signalling axis, and how research on the lymphatic system turned into a hot topic: https://www.nature.com/articles/436456a .

Recombinant Protein Production, CAM Assays, VEGF-D, Transgenic Mice

Sat, 01 Nov 1997 00:00:00 +0000